2017 WHO TRS 1004 Biological Standardization
2017 WHO TRS 1004 Biological Standardization
2017 WHO TRS 1004 Biological Standardization
1004
W H O Te c h n i c a l R e p o r t S e r i e s
and control of vaccines and other biological substances, and the
establishment of international biological reference materials.
Following a brief introduction, the report summarizes a number
1004
of general issues brought to the attention of the Committee. The
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W H O Te c h n i c a l R e p o r t S e r i e s
1 0 0 4
This report contains the collective views of an international group of experts and
does not necessarily represent the decisions or the stated policy of the World Health Organization
WHO Library Cataloguing-in-Publication Data
WHO Expert Committee on Biological Standardization, sixty-seventh report
(WHO technical report series ; no. 1004)
ISBN 978-92-4-121013-3
ISBN (PDF) 978-92-4-069645-7
ISSN 0512-3054
vi
WHO Expert Committee on Biological Standardization
17 to 21 October 2016
Committee members1
Professor K. Cichutek, Paul-Ehrlich-Institut, Langen, Germany (Chair)
Dr J. Epstein, Center for Biologics Evaluation and Research, Food and Drug Administration,
Silver Spring, MD, United States of America (USA) (also Blood Regulators Network
(BRN) representative)
Dr E. Griffiths, Kingston-upon-Thames, England (Rapporteur)
Dr S. Hindawi, Blood Transfusion Services, Jeddah, Saudi Arabia
Mrs T. Jivapaisarnpong, Department of Medical Sciences, Ministry of Public Health,
Nonthaburi, Thailand
Dr H. Klein, National Institutes of Health, Bethesda, MD, the USA (Vice-Chair)
Dr P. Minor, National Institute for Biological Standards and Control, Potters Bar, England
Dr F. Moftah, Ministry of Health and Population, Cairo, Egypt
Mr V.R. Reddy,2 South African National Blood Service, Weltevreden Park, South Africa
Dr L.S. Slamet, Technical Adviser and Consultant to the National Agency of Drug and
Food Control, Jakarta Selatan, Indonesia
Dr P. Strengers, Sanquin, Amsterdam, the Netherlands
Dr Y. Sohn, Ministry of Food and Drug Safety, Chungcheongbuk-do, Republic of Korea
Dr J. Wang, National Institutes for Food and Drug Control, Beijing, China
Temporary advisers
Dr S. Baylis,3 Paul-Ehrlich-Institut, Langen, Germany
Dr M. Gruber, Center for Biologics Evaluation and Research, Food and Drug Administration,
Rockville, MD, the USA
Dr C. Morris, National Institute for Biological Standards and Control, Potters Bar, England
(Rapporteur for the blood products and in vitro diagnostics track)
1
The decisions of the Committee were taken in closed session with only members of the Committee
present. Each Committee member had completed a Declaration of Interests form prior to the meeting.
These were assessed by the WHO Secretariat and no declared interests were considered to be in conflict
with full meeting participation.
2
Unable to attend.
3
Participated via teleconference.
vii
WHO Expert Committee on Biological Standardization Sixty-seventh report
Participants
Dr F. Agbanyo, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa,
Canada (also BRN representative)
Dr N. Almond, National Institute for Biological Standards and Control, Potters Bar, England
Dr L Amsler,5 Swiss Agency for Therapeutic Products, Bern, Switzerland
Dr J. Boyle,6 National Institute for Biological Standards and Control, Potters Bar, England
Dr A. Bristow, National Institute for Biological Standards and Control, Potters Bar, England
Professor D. Calam, Pewsey, England (International Nonproprietary Names (INN) expert)
Dr F. Cano, Agence nationale de sécurité du médicament et des produits de santé,
Lyons, France
WHO Technical Report Series, No. 1004, 2017
4
Participated via teleconference.
5
Participated only in closed BRN session (observer).
6
Participated via teleconference.
7
Participated via teleconference.
viii
WHO Expert Committee on Biological Standardization
Dr B. Cowper,8 National Institute for Biological Standards and Control, Potters Bar, England
Dr R. Dominguez Morales, Ministerio de Salud Pública, Havana, Cuba
Professor S. Efstahiou, National Institute for Biological Standards and Control, Potters Bar,
England
Dr L. Elmgren, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa,
Canada (also BRN representative)
Dr S. Fakhrzadeh, Ministry of Health and Medical Education, Tehran, the Islamic Republic
of Iran
Dr J. Ferguson,9 National Institute for Biological Standards and Control, Potters Bar, England
Dr K. Grant, Therapeutic Goods Administration, Woden, ACT, Australia
Dr E. Gray,10 National Institute for Biological Standards and Control, Potters Bar, England
Dr I. Hamaguchi, National Institute of Infectious Diseases, Tokyo, Japan (also BRN
representative)
Mr J. Hou, National Institutes for Food and Drug Control, Beijing, China
Dr A. Hilger,11 Paul-Ehrlich-Institut, Langen, Germany
Dr C.C. Ilonze, National Agency for Food and Drug Administration and Control, Lagos,
Nigeria
Ms S. Jadoonkittinan, Ministry of Public Health, Nonthaburi, Thailand
Mrs W. Jariyapan, Ministry of Public Health, Nonthaburi, Thailand
Dr H. Jia,12 National Institute for Biological Standards and Control, Potters Bar, England
Dr A. Kato, National Institute of Infectious Diseases, Tokyo, Japan
Dr B. Kim, Ministry of Food and Drug Safety, Chungcheongbuk-do, Republic of Korea
Dr J. Kim, Ministry of Food and Drug Safety, Chungcheongbuk-do, Republic of Korea
Dr T. Kondo,13 Ministry of Health, Labour and Welfare, Tokyo, Japan
Dr C. Lee, Ministry of Food and Drug Safety, Chungcheongbuk-do, Republic of Korea
Dr N. Lelie,14 Scientific Affairs Consultant, Paris, France
8
Participated via teleconference.
9
Participated via teleconference.
10
Participated via teleconference.
11
Participated only in closed BRN session.
12
Participated via teleconference.
13
Participated only in closed BRN session via teleconference.
14
Participated via teleconference.
ix
WHO Expert Committee on Biological Standardization Sixty-seventh report
Dr C. Longstaff,15 National Institute for Biological Standards and Control, Potters Bar,
England
Dr J. Martin,16 National Institute for Biological Standards and Control, Potters Bar, England
Dr F. Mawas,17 National Institute for Biological Standards and Control, Potters Bar, England
Dr H. Meyer, Paul-Ehrlich-Institut, Langen, Germany
Mr H. Mogtari, Food and Drugs Authority Ghana, Accra, Ghana
Ms N.A.M. Nur, National Pharmaceutical Regulatory Agency, Selangor, Malaysia
Dr M. Ochiai, National Institute of Infectious Diseases, Tokyo, Japan
Dr H. Oh, Ministry of Food and Drug Safety, Chungcheongbuk-do, Republic of Korea
Dr I. Osipova, Ministry of Health of the Russian Federation, Moscow, Russian Federation
Dr W. Oualikene-Gonin,18 Agence nationale de sécurité du médicament et des produits
de santé, Saint Denis, France
Dr D. Padley,19 National Institute for Biological Standards and Control, Potters Bar, England
Dr M. Page,20 National Institute for Biological Standards and Control, Potters Bar, England
Dr S. Phumiamorn, Ministry of Public Health, Nonthaburi, Thailand
Ms E.I. Prawahju, National Agency of Drug and Food Control, Jakarta Pusat, Indonesia
Dr S. Prior,21 National Institute for Biological Standards and Control, Potters Bar, England
Dr I. Prosser,22 Therapeutic Goods Administration, Woden, ACT, Australia
Dr S. Raut,23 National Institute for Biological Standards and Control, Potters Bar, England
Dr A. Reissinger,24 Paul-Ehrlich-Institut, Langen, Germany
Dr M. Rios, Center for Biologics Evaluation and Research, Food and Drug Administration,
Silver Spring, MD, the USA25
Ms J. Rodgers, Food and Drugs Authority Ghana, Accra, Ghana
WHO Technical Report Series, No. 1004, 2017
15
Participated via teleconference.
16
Participated via teleconference.
17
Participated via teleconference.
18
Participated only in closed BRN session.
19
Participated via teleconference.
20
Participated via teleconference.
21
Participated via teleconference.
22
Participated only in closed BRN session via teleconference.
23
Participated via teleconference.
24
Participated via teleconference.
25
Participated via teleconference.
x
WHO Expert Committee on Biological Standardization
26
Participated only in closed BRN session.
27
Participated only in closed BRN session via teleconference.
28
Participated via teleconference.
29
Participated via teleconference.
30
Participated via teleconference.
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WHO Expert Committee on Biological Standardization Sixty-seventh report
31
A maximum of two representatives of the Developing Countries Vaccine Manufacturers Network and
two representatives of the International Federation of Pharmaceutical Manufacturers & Associations were
present in the meeting room during discussion of any one agenda item.
32
Participated via teleconference.
33
Participated via teleconference.
34
Participated via teleconference.
xii
WHO Expert Committee on Biological Standardization
WHO Secretariat
Regional Offices
Regional Office for Africa – Dr B. Akanmori
Regional Office for the Eastern Mediterranean – Dr Y. Abdella and Dr H. Langar
Regional Office for South-East Asia – Mr M. Eisenhawer
Regional Office for the Western Pacific – Dr J. Shin
Headquarters
Dr S. Hill, Director, WHO Department of Essential Medicines and Health Products (EMP)
Dr D.J. Wood, Coordinator, Technologies, Standards and Norms (TSN)/EMP (Committee
Secretary)
Dr J. Fournier-Caruana, Prequalification Team (PQT)/EMP
Dr K. Gao, TSN/EMP
Dr J. Hansen, TSN/EMP
Dr J. Hombach, Department of Immunization, Vaccines & Biologicals (IVB)/Initiative for
Vaccine Research (IVR)
Dr H-N Kang TSN/EMP
Dr I. Knezevic TSN/EMP (Lead for the vaccines and biotherapeutics track)
xiii
WHO Expert Committee on Biological Standardization Sixty-seventh report
Dr D. Lei TSN/EMP
Dr R. Meurant, PQT/EMP
Dr M. Nuebling, TSN/EMP (Lead for the blood products and in vitro diagnostics track)
Ms I. Prat, PQT/EMP
Ms C.A. Rodriguez-Hernandez, PQT/EMP
Dr H. Schmidt, TSN/EMP
Dr I. Shin, TSN/EMP
Dr T. Zhou, TSN/EMP
WHO Technical Report Series, No. 1004, 2017
xiv
Abbreviations
Ab antibody
Ag antigen
anti-HBc antibodies to hepatitis B core protein
anti-HBs antibodies to hepatitis B surface antigen
ASfBT Africa Society for Blood Transfusion
BGTD Biologics and Genetic Therapies Directorate
BRN WHO Blood Regulators Network
CBER Center for Biologics Evaluation and Research
CEG Core Expert Group
CHIKV chikungunya virus
CTP cell therapy product
DENV dengue virus
DNA deoxyribonucleic acid
DTP diphtheria, tetanus and pertussis
EBOV Ebola virus
ECSPP WHO Expert Committee on Specifications for Pharmaceutical
Preparations
EDQM European Directorate for the Quality of Medicines & HealthCare
EGFR epidermal growth factor receptor
ELISA enzyme-linked immunosorbent assay
EMA European Medicines Agency
EMP WHO Department of Essential Medicines and Health Products
EUAL WHO emergency use assessment and listing (procedure)
EV enterovirus
EVD Ebola virus disease
EQA external quality assurance
FDA Food and Drugs Authority (Ghana)
FV blood coagulation factor V
xv
WHO Expert Committee on Biological Standardization Sixty-seventh report
xviii
1. Introduction
The WHO Expert Committee on Biological Standardization met in Geneva
from 17 to 21 October 2016. The meeting was opened on behalf of the Director-
General of WHO by Dr Suzanne Hill, the recently appointed Director of Essential
Medicines and Health Products (EMP). Dr Hill welcomed the Committee,
meeting participants and observers. She informed the meeting of several staff
changes in the Department over the previous year, with a number of further
senior WHO staff changes expected in the near future. This would include
the Secretary to the WHO Expert Committee on Biological Standardization,
Dr David Wood, who would be retiring in early 2017.
Dr Hill referred to the United Nations overarching strategic
direction entitled “Transforming our world: The 2030 Agenda for Sustainable
Development” 1 and indicated that this was being translated into a new vision for
EMP. One new initiative would be a greater focus on access to biotherapeutics.
It is envisaged that by 2030, biological substances will be used more widely than
at present and ensuring sustainable access to biotherapeutics of assured quality
for public health-care systems will be a key challenge for all countries, rich and
poor alike. Dr Hill emphasized that the development and adoption of norms
and standards to regulate the quality, safety and efficacy of medical products
and guide their cost-effective use would be a critical foundation on which future
aspirations would be built. A global approach to this core normative work is
facilitated by the coordinated efforts of WHO Expert Committees and the vital
support of WHO collaborating centres (WHO CCs) and partner organizations.
Dr Hill reminded meeting participants that the Committee had a
mandate, enunciated in the WHO Constitution, to develop, establish and
promote international standards for biological products. In addition to
supporting the development and use of biological medicines, other goals now
needed to be considered – specifically, promoting access to essential medicines
and regulatory strengthening. Furthermore, the world of regulatory science was
changing, particularly as new products came to the market, and there was an
expectation concerning the role of WHO in providing regulatory guidance and
promoting regulatory strengthening. Norms and standards underpin and reflect
these expectations but need to be regularly reviewed to ensure they reflect the
best regulatory science. The convergence of norms and standards internationally
is recognized as a key driver in addressing these needs.
After norms and standards have been established there was then a
need for proactive technical support from WHO to its Member States in order
17 February 2017).
1
WHO Expert Committee on Biological Standardization Sixty-seventh report
Substances was also taking place. Dr Wood then introduced the members of the
2016 Expert Committee on Biological Standardization, and highlighted the new
requirement initiated in 2015 that biographical summaries of all the members
must be posted for public review and comment prior to the meeting. All
biographical summaries had been posted and no comments had been received.
Dr Wood then outlined the organization of the meeting and the major issues
to be discussed. Declarations of Interests made by members of the Committee,
Temporary Advisers and participants were then presented.2 Following prior
evaluation, WHO had concluded that none of the declarations made constituted
a significant conflict of interest, and that the individuals concerned would be
allowed to participate fully in the meeting.
Following participant introductions, the Committee adopted the
proposed agenda (WHO/BS/2016.2303 Add.1).
3
2. General
2.1 Current directions
2.1.1 Strategic directions in biological standardization: WHO priorities
Dr Wood reminded the Committee of the core activities of WHO and that its
Constitution required it ...to develop, establish and promote international standards
with respect to biological and pharmaceutical products. WHO had been doing
this for over 60 years through a programme which included the development
of global written standards, global measurement standards and International
Nonproprietary Names (INN). In the field of biological substances there were
now over 70 WHO written standards and 300 reference preparations, all of
which make a significant contribution to global public health. Indeed, the first
international biological reference preparation, for insulin, had been established
in 1925 under the auspices of the League of Nations. Current global public health
priorities include responding to public health emergencies of international
concern, access to biotherapeutics and the strengthening of regulatory systems
– the latter two being supported by two World Health Assembly resolutions:
one on biotherapeutic products (WHA67.21, 2014) and the other on
regulatory systems strengthening (WHA67.20, 2014). Resolution WHA67.21
requests WHO to support Member States in the regulation of biotherapeutic
products, including similar biotherapeutic products (SBPs). In particular, the
Resolution requested WHO to convene the Expert Committee on Biological
Standardization in order to update its 2009 Guidelines in this area, taking
into account technological advances in the characterization of biotherapeutic
products and considering national regulatory needs and capacities. WHO had
recently reported on progress in this area to its Executive Board and to the
sixty-ninth World Health Assembly in May 2016.
As part of this programme, the Committee had adopted key written
WHO Technical Report Series, No. 1004, 2017
need to consider very carefully the potential use and extent of applicability of
such reference preparations.
In considering the workload in the biologicals field, Dr Wood raised
the issues of the increasingly packed agendas of meetings of the Committee, the
difficulties in minimizing scheduling conflicts in the two-track system and the
insufficient time allocated for discussing strategic issues. Various proposals
were being considered to optimize the use of Committee time during face-to-
face meetings, such as holding meetings biannually, and the introduction of
more pre-meeting technical discussion of proposals for reference preparations.
Discussion at the face-to-face meetings could then be reserved for the more
complex proposals or those that were precedent-setting. Increasing the
inputs from WHO CC networks (see sections 2.2.2 and 2.2.3 below) was also
under consideration. Currently, WHO CC networks exist for vaccines and for
blood products and in vitro diagnostics (IVDs) but not for biotherapeutics.
Dr Wood also added that the WHO Secretariat is currently understaffed but
that additional staffing was being sought through secondments. Linkages with
other Expert Committees and Expert Groups were also considered important,
for example in developing both biological and chemical reference preparations
for biotherapeutic medicines, along with efforts to raise the visibility of the
work carried out in the area of biological standardization.
The Committee thanked Dr Wood for his overview and expressed
support for the focus and priorities outlined. The Committee drew attention to
standardization needs in potential new areas of work, such as vaccine platforms,
companion diagnostics and cell therapies, and highlighted the need to consider
where such activities might fit into the WHO programme of work. Such
consideration should include a careful review of the scope of the Committee,
which currently includes vaccines, biotherapeutics, blood products and IVDs.
The Committee also agreed that there was a need to improve the visibility of
the WHO biological standardization programme and its key role in supporting
global public health. This should involve not only reporting on the work of the
Committee in the scientific media but also keeping medical prescribers and
practitioners aware of the WHO biological standardization programme and its
significance in assuring the quality, safety and efficacy of biological medicines.
Indeed, the Committee considered that improving visibility and explaining the
crucial need for biological standardization should start during the education of
medical and pharmaceutical science students.
In: WHO Expert Committee on Biological Standardization: sixty-sixth report. Geneva: World Health
3
Organization; 2016: section 8.1.1 (WHO Technical Report Series, No. 999; http://apps.who.int/iris/bitstre
am/10665/208900/1/9789240695634-eng.pdf?ua=1, accessed 17 February 2017).
7
WHO Expert Committee on Biological Standardization Sixty-seventh report
now being presented to the Committee for adoption (see section 3.2.6 below).
Dr Nuebling indicated that if antivenom assessment is to become a sustainable
process in the future then funding issues will need to be addressed. Planned
next steps include the completion of first-round assessment based on laboratory
testing of promising products, funded by Médecins Sans Frontières (MSF), and
inspections of manufacturing sites. A side event on antivenoms took place at
the 2016 World Health Assembly, initiated by Costa Rica and supported by
17 Member States, with the intention of raising awareness and potential donor
interest in this important public health issue. Moreover, snake-bites will be
proposed for re-entry into the WHO List of Neglected Tropical Diseases and
the topic has been proposed as an agenda item of the WHO Executive Board.
There are also plans to promote snake-bite prevention and treatment initiatives,
including antivenom technology transfer, early case management and research.
Due to the 2015–2016 ZIKV epidemic in the Americas, and the
suspected association in Brazil between ZIKV infection in pregnant women and
microcephaly in newborns, WHO guidance on blood donations was urgently
required. Following close cooperation between EMP, the WHO Department of
Service Delivery Systems and the WHO Regional Office for the Americas, with
support from the WHO Blood Regulators Network (BRN), a WHO guidance
document on blood collection was published in February 2016. This document
advises on situations with and without active ZIKV transmission and evaluates
potential measures such as donor deferral, quarantining of blood components,
pathogen inactivation and testing. There was also an urgent need for reference
materials in this area, and projects to develop reference preparations for ZIKV
IVDs had therefore been initiated. This resulted in the rapid development of a
candidate WHO standard for ZIKV RNA to be considered for establishment
by the Committee this year (see section 7.1.1 below). Problems had been
encountered however in obtaining materials for serology standards.
Dr Nuebling also reported on the progress of the Achilles project for
improving access to safe blood products through local production and technology
transfer in blood establishments. Indonesia had been chosen as a pilot country
for the Achilles project. Project activities included the evaluation of IVDs related
to blood safety since many tests were available but their performance data were
not usually assessed. A workshop was held in Jakarta in August 2016 to review
current practices. Workshop participants included representatives of Ministry of
Health departments, the hepatitis surveillance programme, the Indonesian Red
Cross and the IVD laboratory of the Indonesian Centers for Disease Control.
The main aim was to evaluate > 40 different rapid diagnostic tests (RDTs) for
the detection of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections.
RDTs are still used for blood screening for 15% of the national blood supply and
are the main surveillance tool in the country. Protocols for evaluating all HBV
and HCV RDTs were reviewed with an initial focus on sensitivity.
9
WHO Expert Committee on Biological Standardization Sixty-seventh report
2.2 Reports
2.2.1 Report from the WHO Blood Regulators Network
WHO Technical Report Series, No. 1004, 2017
Dr Christian Schaerer reported on the activities of the BRN during the past year.
Following its face-to-face meeting during the previous Committee meeting in
October 2015, five teleconferences had been held. There had also been several
changes in BRN member representatives as well as a minor revision of the
BRN Terms of Reference. This now allowed “alternate” representatives to be
eligible candidates for the BRN Chair. Dr Schaerer himself had been elected
BRN Chair for a second term, until October 2017.
BRN activities during 2015–2016 had included discussion of the ongoing
revision of the WHO NRA assessment tools, and of the draft WHO Guidelines
on estimation of residual risk of HIV, HBV or HCV infections via cellular blood
components and plasma, proposed for adoption at this meeting (see section
3.2.4 below). Discussions were also held on the first results of clinical trials
10
General
that the Terms of Reference make it clear that candidates should be experienced
blood regulating authorities. It was therefore suggested that the BRN develop
mechanisms for observing and taking into consideration the perspectives of
other countries. Dr Schaerer referred to already existing processes for such
interactions, such as the consultation phases for guidance documents.
It was also suggested that the BRN, in addition to responding to new
demands or emergencies, should also strengthen its support for the educating
and training of regulators in NRAs less experienced in blood regulation. It was
again emphasized that BRN members, despite limited capacities, were already
active in this endeavour through workshops in developing countries, for example
in Africa.
therefore been proposed that a “Core Expert Group” (CEG) from the network
be formed. As new measurement standards have strategic considerations (in
addition to scientific issues) it was felt that these should remain the responsibility
The network currently consists of the following eight WHOCCs: (a) National Institute for Biological
4
Standards and Control (NIBSC), Medicines and Healthcare products Regulatory Agency, England;
(b) Center for Biologics Evaluation and Research (CBER), Food and Drug Administration, the USA;
(c) Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Diseases
(NIID), Japan; (d) Immunobiology and Biochemistry Group, Office of Laboratories & Scientific Services,
Therapeutic Goods Administration (TGA), Australia; (e) National Institute of Food and Drug Safety
Evaluation (NIFDS), Ministry of Food and Drug Safety (MFDS), Republic of Korea; (f ) Biologics and Genetic
Therapies Directorate (BGTD), Health Canada, Canada; (g) Institute for Biological Product Control of the
National Institutes for Food and Drug Control (NIFDC), China; and (h) Division of Virology, Paul-Ehrlich-
Institut (PEI), Germany.
12
General
Committee and to discuss new project areas. Dr Morris summarized the agenda
topics discussed, which in the case of two items – namely the dengue virus
(DENV) RNA reference reagent and the anti-CMV collaborative studies – had
led to a re‑evaluation of data and alteration of the final proposals made to
the Committee.
Comments from users of current standards, which are not normally
considered at routine meetings of the Committee, had also been discussed.
Dr Morris gave the example of the B19 DNA standard, explaining that some
users of one specific test (Roche DPX) had reported under-quantification of the
International Standard. On further investigation a single base-pair mismatch
was identified in the current standard compared to that of the manufacturer’s
primers/probes. Despite calls for the revision of the assigned IU of the standard,
it was agreed by the network, and by the delegates of the annual Standardisation
of Genome Amplification Techniques (group) (SoGAT) meeting, that the unit
should remain. Difficulties were also reported in reconstituting the current HCV
standard. This standard had been produced from a donation that on freeze-
thawing showed signs of insoluble lipid particles. These also appeared following
reconstitution. Users have now been advised to ensure thorough mixing
immediately prior to use to ensure that particulate matter is also incorporated
into the extraction.
Dr Morris reported that the WebEx meetings were considered overall
to have been a valuable addition to the network meeting calendar, allowing
pertinent issues to drive the agenda in a timely way while assisting in the
streamlining of the main process of standards approval by the Committee.
During discussion, it was suggested that where a project is considered
straightforward, a one-page summary could be submitted to the Committee for
its review and endorsement without requirement for a presentation of the project
itself. Where a project was considered more complex and issues remained to be
addressed, a two-page proposal could be submitted and the Committee could
WHO Technical Report Series, No. 1004, 2017
also seek further clarification in the form of a presentation. However, it was also
made clear that the work of the WHO CCs could not supplant the role of the
Committee in establishing standards and endorsing new work. The Committee
thanked Dr Morris for her presentation and agreed that network meetings of the
kind outlined should be pursued and further explored as a means of facilitating
the work of the Committee.
testing and participation in on-site inspections. The PEI was also involved in
activities related to: (a) the ongoing ZIKV public health emergency, including
the development of a WHO international standard for ZIKV RNA for NAT-
based assays (see section 7.1.1 below); (b) the WHO emergency use assessment
and listing (EUAL) procedure for ZIKV diagnostics; and (c) the safe use of
plasma-derived medicinal products (PDMPs). With regard to the latter, the
bloodborne transmission of ZIKV had raised the issue of PDMP safety and
PEI researchers therefore investigated the effectiveness of the most commonly
used virus-reduction/inactivation methodologies. Pasteurization and solvent/
detergent treatment both led to the rapid inactivation of ZIKV. Retentive virus
filtration was also shown to be effective, with ZIKV infectivity removed by filters
with nominal pore sizes ≤ 40 nm. Dr Meyer also reported that immunoglobulins
sourced in Europe or the USA had failed to neutralize ZIKV.
Dr Meyer acknowledged the excellent cooperation of its partners in the
two WHO CC networks in which it was involved, and highlighted PEI support
for the implementation of documents issued by the BRN. Topics highlighted for
future consideration by the Committee included: (a) considerations in advancing
the scientific evaluation of convalescent plasma collection and use beyond the
Ebola outbreak; (b) implementation of the assessment criteria for national blood
regulatory systems (which had already been applied in Ghana); (c) assessment
of the significance of hepatitis E virus for blood safety; (d) the quality and safety
standards for haematopoietic stem cells; and (e) the role of the Committee in
ensuring sustainable vaccine supply (see section 2.4.4 below). The PEI also
supported a proposal to facilitate the work of the Committee by streamlining the
process for evaluating new projects in the area of vaccines and biotherapeutics,
including the establishment of a process for project prioritization.
It was also reported that the PEI was providing support for two health-
system strengthening and capacity-building efforts. The first involved the
establishment of bilateral interactions with Ghana and Liberia to strengthen their
WHO Technical Report Series, No. 1004, 2017
the proposed establishment of a WHO international standard for ZIKV RNA for
NAT-based assays.
Center for Biologics Evaluation and Research (CBER), Silver Spring, MD, the USA
Dr Jay Epstein informed the Committee of recent and current developments at
CBER, including the appointment of Dr Peter Marks as Director following the
retirement of Dr Karen Midthun. Organizational restructuring had resulted in
three product line offices: the Office of Blood Research and Review, the Office of
Vaccines Research and Review and the Office of Tissues and Advanced Therapies.
Products regulated by the latter will include all purified and recombinant versions
of therapeutic proteins for use in haematology, as well as antivenins. CBER had
also been redesignated as a WHO CC for the period 2016–2020 and an umbrella
agreement had been put in place with WHO on vaccines, blood and blood
products, relevant IVDs and cell and tissue therapies. This agreement will support
the development of norms and standards, regulatory systems strengthening, the
WHO prequalification programme, product safety and vigilance, and regulatory
science in order to increase access to safe and effective biological products.
Dr Epstein outlined new United States Food and Drug Administration
NAT-testing policies with regards to blood donations (which are intended
to reduce the transfusion risk from ZIKV) as well as revised donor-deferral
criteria for HIV risk which permit men who have sex with men to donate
blood under certain circumstances. A Transfusion Transmissible Infections
Monitoring System had also been established to assess infectious disease risks
based on marker rates, incidence and risk factors in blood donors. Dr Epstein
also described CBER’s global involvement with other WHO CCs in the
standardization of plasma-derived coagulation factors, and in the development
of reference reagents and panels for the standardization of assays for transfusion-
transmitted infectious agents such as DENV, CHIKV, West Nile virus, HIV and
ZIKV, as well as for Babesia microti antibodies.
Recent CBER activities related to vaccines included participation in
an international study to evaluate new methods for measuring the potency
of inactivated influenza virus vaccines compared with the traditional single
radial immunodiffusion assay. Following completion, study results indicated
that despite the general feasibility of all alternative assays, additional studies
would be needed to identify the most promising ones for further development
and possible implementation. A second collaborative study was now under
development and scheduled for early 2017. An international study to evaluate
the inter-laboratory variability of influenza virus serological assays had also
been conducted. Coordinated by the Consortium for the Standardization of
Influenza Seroepidemiology, this study compared the inter-laboratory variability
of the standardized haemagglutination inhibition (HI) and microneutralization
19
WHO Expert Committee on Biological Standardization Sixty-seventh report
(MN) assay protocols, with CBER performing the HI, MN and pseudotype
neutralization assays. Twenty laboratories provided data, and statistical analysis
of the results was ongoing at the NIBSC. Preparations for dealing with new
inactivated poliomyelitis vaccines (IPVs) based on Sabin vaccine strains instead
of conventional IPVs were also under way. Conventional IPV and Sabin IPV
differ antigenically and the current D-antigen potency ELISAs for conventional
IPV may not be suitable for Sabin IPV vaccines. A PATH-sponsored collaboration
with the Lankenau Institute of Medical Research to evaluate human monoclonal
antibodies in new D-antigen potency ELISAs has therefore been initiated. In
addition, CBER is supporting the development of new vaccine technologies
by participating in studies led by the Advanced Virus Detection Technologies
Interest Group (which is supported by the Parenteral Drug Association) to
develop next-generation sequencing controls for detecting adventitious viruses
in biological substances. CBER was responsible for preparing the large well-
characterized virus stocks to be used as controls in spiking studies for the
evaluation and standardization of next-generation sequencing platforms.
The Committee thanked Dr Epstein for his presentation and asked
whether the standardization materials being developed for next-generation
sequencing would be applicable across all available platforms. Dr Epstein
confirmed that this was the intention.
WHO Global Model Regulatory Framework for Medical Devices including IVD medical devices. In: WHO
5
Expert Committee on Specifications for Pharmaceutical Preparations: fifty-first report. Geneva: World
Health Organization; 2017: Annex 4 (WHO Technical Report Series, No. 1003).
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WHO Expert Committee on Biological Standardization Sixty-seventh report
22
General
laboratories. Microbiological assays, on the other hand, measure the total activity
of antibiotics, integrating all moieties that contribute to the antibiotic effect.
At the 2009 meeting of the ECSPP the decision was taken to replace
microbiological assays, where possible and appropriate, by physicochemical
methods (in particular, chromatographic methods) in The International
Pharmacopoeia. The Committee was informed that this transition from
microbiological to physicochemical assays had been largely completed for single-
component antibiotics. However, for multicomponent compounds the use of
physicochemical methods often poses a challenge as the total antimicrobiological
response of these substances is not only a function of their concentration but
also of their composition.
Capreomycin is a mixture of four structurally related compounds
(capreomycin IA, IB, IIA and IIB) with different individual activities. In The
International Pharmacopoeia monograph on capreomycin sulfate the active
substance is defined on a mass basis, whereas capreomycin is assayed using
high-performance liquid chromatography which discriminates between IA,
IB, IIA and IIB. In 1969, the activities of these four main components were
determined by a manufacturer of capreomycin. The results indicated that there
was a significant difference between the activities of components IA and IB
and between I and II. The United States, Indian and Chinese pharmacopoeias
all have similar requirements regarding the composition of capreomycin
sulfate but in these pharmacopoeias the capreomycin content is determined
using microbiological methods. Although The International Pharmacopoeia
limits capreomycin II content to a maximum of 10%, the ratio between
capreomycin IA and IB is not defined. There is therefore at least a theoretical
possibility that capreomycin samples with significant differences in their IA
and IB concentrations comply with the requirements of The International
Pharmacopoeia, but may be determined as sub- or super-potent when analysed
according the United States, Indian or Chinese pharmacopoeias.
The issue had been referred to the Committee and to the ECSPP for
advice as to whether a revision of the current International Pharmacopoeia
monograph is recommended. Prior to such an amendment, it was proposed that
the secretariat of The International Pharmacopoeia should contact manufacturers
of capreomycin sulfate and powders for injection to request information
regarding the composition of their products – as determined for example by
the chromatographic methods described in The International Pharmacopoeia.
This information would then be evaluated by a group of experts to see whether
amendments to the monograph were appropriate. Such amendments could
include the provision of a specification for the ratio between capreomycin IA
and IB and/or the establishment of a correlation between the results of the
23
WHO Expert Committee on Biological Standardization Sixty-seventh report
volume should be used only as an exceptional response where there was a large
disease outbreak and vaccine shortage – and not in routine immunization.
The WHO SAGE advice was based on limited clinical studies of
fractional dose administration. While the data support the recommendation,
important data gaps remained – such as on vaccination responses among
children and immunocompromised populations to fractional doses and on the
duration of immunity. The Committee was reminded that there were currently
only four WHO-prequalified YF vaccines (all live-attenuated) which differ in
their properties. As batch records for these vaccines have shown excess potency
(albeit with some variability) there was potential scope for reducing the dose
required to achieve an acceptable seroconversion threshold. However, the
available data are based mainly on product from one manufacturer and the
long-term duration of immunity beyond one year following a fractional dose
24
General
6
Recommendations to assure the quality, safety and efficacy of live attenuated yellow fever vaccines. In:
WHO Expert Committee on Biological Standardization: sixty-first report. Geneva: World Health Organization;
2013: Annex 5 (WHO Technical Report Series, No. 978).
7
2014 Ebola virus disease outbreak and follow-up to the Special Session of the Executive Board on Ebola.
Sixty-eighth World Health Assembly, Geneva, 18–26 May 2015. Geneva: World Health Organization; 2015.
Agenda item 16.1 (http://apps.who.int/gb/ebwha/pdf_files/WHA68/A68_ACONF5-en.pdf, accessed 19
February 2017).
25
WHO Expert Committee on Biological Standardization Sixty-seventh report
adapted to the scientific, logistic and social challenges specific to epidemics. Four
principles underpin the project: (a) an inclusive process with a clear mandate and
milestones; (b) building on the efforts of others; (c) a collaborative effort with
affected Member States at the core of public health emergency responses; and
(d) a process driven by scientific knowledge. The R&D Blueprint aims to reduce
the time between the declaration of an international public health emergency
and the availability of effective tests, vaccines and medicines that can be used to
save lives and avert a crisis.
The R&D Blueprint has three work streams: (a) improving coordination
and fostering an enabling environment – for example, by building effective
governance and coordination frameworks; (b) accelerating the research and
development processes – for example, by assessing epidemic threats and
defining priority pathogens – and then in a second step by developing R&D
roadmaps to accelerate evaluation of diagnostics, therapeutics and vaccines;
and (c) developing new norms and standards adapted to the epidemic context
– for example, supporting expansion of capacity to implement adequate study
designs, developing guidance and tools to frame collaborations and exchanges,
and anticipating the evidence needed to inform regulatory review and policy
development. With regard to research strategy aspects, the Committee was
informed that a recent call for platform technologies for vaccine production,
medicines and diagnostics had been made by WHO. Forty-five proposals
were subsequently submitted, of which six were selected for presentation to
potential funders.
Priority pathogens and diseases listed in 2016 were: Crimean Congo
haemorrhagic fever, filovirus disease (EBOV and Marburg virus diseases), Lassa
fever, highly pathogenic emerging coronaviruses relevant to humans (MERS-
CoV and severe acute respiratory syndrome), Nipah, Rift Valley fever and
ZIKV. Two other diseases listed as serious are chikungunya and severe fever
with thrombocytopenia syndrome. The list will be reviewed annually or when
WHO Technical Report Series, No. 1004, 2017
a new disease emerges. It was intended that R&D roadmaps for all priority
pathogens would be developed to guide the R&D response to large-scale public
health challenges, with such a roadmap having been developed for MERS-
CoV as a test case. The R&D Blueprint was also intended to link together other
international efforts, such as The Coalition for Epidemic Product Innovation
established following the Annual Meeting of the World Economic Forum in
Davos in January 2016.
The Committee welcomed this WHO initiative and noted that
considerable efforts were already under way in the area of norms and standards.
Guidelines on the quality, safety and efficacy of Ebola vaccines were being
developed (see section 3.5.6 below) – the principles of which could inform the
evaluation of similar vaccines against other priority pathogens. In addition, the
WHO CCs at NIBSC and PEI had developed international reference reagents
26
General
for pathogens with epidemic potential, including standards for ZIKV RNA and
for EBOV antigen and antibodies. However, the Committee also agreed that
more remained to be done and there were still many challenges to be resolved.
One problem was that of obtaining clinical material from priority pathogens
with which to produce international reference materials. Although efforts were
now under way to facilitate the sharing of clinical material, sending biological
materials across national and state borders, especially convalescent sera and
infectious materials, was often problematic. The Committee recommended
that WHO should play a role in facilitating procedures and ensuring that the
pathways for providing material to the relevant WHO CC were made clear.
The Committee also raised the possibility of using blood donations
as a source material for research. Despite challenges in ensuring informed
consent, this had been shown to be a viable option – for example, for following
virus evolution using next-generation sequencing technologies or for better
understanding epidemics with respect to pre-symptomatic patients via screening
programmes. The possibility of using material from deferred blood donations
to prepare reagents might also be considered.
The question was raised as to whether the use of convalescent plasma
as a treatment option could realistically benefit from a platform approach given
that in the case of the Ebola epidemic several issues could not be addressed due
to a lack of time and infrastructure. Such issues included determining the ideal
time point after infection or during convalescence to procure plasma with high
neutralizing titres.
27
3. International Recommendations, Guidelines and
other matters related to the manufacture, quality
control and evaluation of biological substances
All WHO Recommendations, Guidelines and guidance documents adopted at
the meeting are included in Annex 1, which provides an updated listing of all
current WHO Recommendations, Guidelines and other documents related to
the manufacture, quality control and evaluation of biological substances used
in medicine.
data set for licensure. However, because of the structural complexity of mAbs,
comparability studies between a candidate biosimilar mAb and a reference
product mAb are challenging for both developers and regulators.
In 2014, the World Health Assembly adopted a resolution (WHA67.12)
on Access to biotherapeutic products, including similar biotherapeutic products,
and ensuring their quality, safety and efficacy. This resolution requested WHO
through its Expert Committee on Biological Standardization to update the 2009
Guidelines, taking into account technological advances in the characterization
of biotherapeutic products, and considering national regulatory needs and
capacities. A corresponding request was then made at the 16th ICDRA. In
response, WHO organized an informal consultation in 2015 to review the 2009
Guidelines and to consider ways of improving its guidance in this important
pharmaceutical sector. Participants included NRAs and national control
28
International Recommendations, Guidelines and other matters
laboratories (NCLs) from 26 countries in the six WHO regions, together with
developed and developing country manufacturers’ associations and individual
manufacturers. It was concluded there was no need to revise the overarching
2009 WHO Guidelines since the evaluation principles described still applied
and were valuable in facilitating the convergence of SBP requirements globally.
However, it was also agreed that, because of their complexity, there was a need for
additional WHO guidance on the evaluation of biosimilar mAbs.
Consequently, class-specific guidance on special considerations for the
evaluation of mAbs developed as SBPs was prepared and subjected to global
public consultation. The resulting draft Guidelines (WHO/BS/2016.2290)
covered rDNA-derived biosimilar mAbs, as well as mAb-derived fragments and
Fc fusion proteins, used in the treatment of human diseases. These Guidelines
are intended to be read in conjunction with the existing WHO Guidelines on
the quality, safety and efficacy of biotherapeutic protein products prepared
by recombinant DNA technology and the WHO Guidelines on evaluation of
similar biotherapeutic products (SBPs), and are intended to complement existing
relevant regulatory documents from other bodies.
The Committee reviewed the small number of comments that had
been received during a final round of public consultation. After agreeing
upon a number of further amendments to improve the clarity of the text, the
Committee recommended that the WHO Guidelines be adopted and annexed
to its report (Annex 2).
workforce currently dedicated to blood at the Ghana FDA (two full-time staff)
synergies achieved through interaction with other relevant Ghana FDA units
are imperative. In order to understand the status quo, the Ghana FDA had
previously visited more than 50 blood facilities across the country.
The in-country assessment identified areas for improvement and led to
several recommendations. These included making good use of already existing
structures for pharmaceutical regulation and generating synergies with additional
departments, such as those responsible for enforcement. However, blood-
specific training is also necessary, especially in relation to facility inspection and
haemovigilance. For blood-screening IVDs, a reliance on approvals made by
other NRAs or via WHO prequalification should be considered. If IVD testing
is to be performed at the Ghana FDA then the use of regional specimens for
test panels is recommended, rather than simply repeating the tests performed
by manufacturers.
The assessment report, and associated recommendations, were well
received by the Ghana FDA and were used during negotiations with the Ghana
Ministry of Health for further support. Both training (potentially in the form
of twinning projects) and funding will be essential in supporting the currently
understaffed but highly motivated team at Ghana FDA. WHO plans to follow up
with a reassessment after a certain time, but this was dependent upon funding.
Other countries that had expressed an interest in undergoing an external WHO
BRN assessment were Kenya and Zambia.
The Committee noted the assessment report and requested to be kept
informed of future assessments and their outcomes.
products currently being imported from abroad. Whole blood or fresh frozen
plasma is mostly used in place of PDMPs, which both increases the risk of
transfusion-transmitted infections and prolongs treatment. Only South Africa
manufactures products through the fractionation of plasma collected in the
country, and also exports surplus products to neighbouring countries.
During a presentation made to the Committee on behalf of the Africa
Society for Blood Transfusion (ASfBT), an action plan was outlined for possible
adoption by WHO as a set of concrete next steps to further advance the WHO
Achilles project on improving access to safe blood products through local
production and technology transfer in blood establishments. As proposed by
the ASfBT, this action plan for Southern Africa aimed to increase the plasma
supply and to improve access to PDMPs in Angola, Botswana, Lesotho, Malawi,
Mozambique, Namibia, South Africa, Swaziland, Zambia and Zimbabwe.
30
International Recommendations, Guidelines and other matters
(GPP) as used for blood components was raised. As use of the latter term in
this context was in line with other relevant resources and with European Union
terminology, its addition to the Guidelines was accepted. Other discussion topics
raised included the requirement for obtaining specific consent for research, and
the possibility of using accreditation and monitoring systems instead of NRAs
for blood systems. With respect to the latter, it was noted that governmental
NRAs are considered essential for the oversight of blood establishments,
whereas accreditation, despite its potential in enhancing the quality of blood
establishments, was generally voluntary and should only be viewed as a
supplemental system. Although there was also some discussion of the concept
of national self-sufficiency margins defined in terms of annual blood units per
population size, it was decided that all issues pertaining to blood supply lie
outside the scope of the Guidelines.
32
International Recommendations, Guidelines and other matters
trials and products both now in high-income countries and, in the future, in
low- and middle-income countries. The long-term objective would then be
to help improve access to gene therapies of assured quality, safety and efficacy
by 2030.
The clinical development of gene therapies – which are at a more
advanced stage than cellular therapies – began over 20 years ago. Some of the
early clinical trials involved gene therapy vectors which caused leukaemia in
some immunodeficient children through unwanted insertional mutagenesis.
More recent trials, which used different vectors considered to carry less risk of
such adverse events, have led to more encouraging results and to a resurgence of
optimism about these products. The first retroviral gene therapy to treat the rare
inherited disease adenosine deaminase-severe combined immunodeficiency
(ADA-SCID) is now licensed in Europe. Tools to minimize future risk and
38
International Recommendations, Guidelines and other matters
maximize potential benefits would be beneficial and would help realize the
public health benefits of this technology.
Reference preparations would potentially be used by product developers
and manufacturers of gene therapies to calibrate the gene dosage in clinical
trials and thus assist NRAs in making decisions on clinical trial (and eventually
product) approvals. It was also foreseen that hospital diagnostic laboratories
would require tools to help monitor gene therapy patients and the outcomes
of the therapies. The impact of standardization could be the assurance that the
same virus vector is applied under standardized conditions and/or that potential
integration into chromosomes is determined in a standardized way. The use of
reference preparations would also be expected to improve comparability between
clinical studies.
In 2002, WHO established a Clinical Gene Transfer Monitoring Group
which met in 2002 and 2003 and then reported to the Committee. At that time
there was no consensus on the most appropriate approach to standardizing
gene therapy vectors and as a result no WHO reference preparations have yet
been established. However, WHO reference preparations have been established
for genetic tests and the methodology proposed to develop standards for gene
therapy vectors would build upon experience with the genomic reference
materials. In 2005, the WHO INN Expert Group established a naming policy
for gene therapy products and has, to date, provided names for 24 candidate
products. Analysis of the applications being made to the WHO INN programme
provides a means of identifying trends in the field and the potential need for
biological standards. NIBSC has identified the standardization of gene copy
number for candidate products being tested in human clinical trials as a
sufficiently mature area of work to potentially benefit from the establishment
and maintenance of WHO biological standards. Based on a scientific workshop
convened by NIBSC in June 2016 a proposal has been made to develop a
lentiviral vector copy number standard (see section 6.1.1 below). Lentiviral
vectors are emerging as a platform technology for gene therapies. If other such
platform technologies are developed, and if WHO initiates work on reference
preparations, then WHO would need to consider establishing appropriate
additional biological standards for these other platform technologies. The
National Institute for Standards and Technologies (NIST) has also organized
workshops on standardization in the field of advanced therapy medicinal
products but there is no overlap with potential WHO projects on gene therapy.
The Committee agreed that the development of reference preparations
for gene therapy products should be explored further and considered the
proposed development of a lentivirus reference to be a good model exercise.
It also noted that the need for a corresponding written standard would be
considered at a future meeting.
39
WHO Expert Committee on Biological Standardization Sixty-seventh report
draft was prepared and more widely circulated among relevant parties. Following
consideration of this document by the Committee in 2015, incorporation of the
points raised, and additional stakeholder discussions the proposed document
(WHO/BS/2016.2284) was submitted to the Committee for adoption.
The Committee was informed that the document was intended to provide
practical guidance on the preparation and calibration of secondary standards,
and would be of use to bodies that prepare and establish secondary standards,
IVD manufacturers, and providers of external quality assurance or proficiency
testing programmes, as well as to other laboratories using reference materials
for NAT-based and serological infectious disease assays. Due to their inherent
complexity – for example, inter-individual differences in antibody response and
inter-assay design differences – the scope of the document did not cover materials
40
International Recommendations, Guidelines and other matters
used for antibody detection methods. The Committee was then provided with an
overview of the structure and content of the proposed document.
The Committee considered that the document as a whole provided
a better understanding of calibration than part B of the current WHO
Recommendations in this area.8 It was also agreed that the document should
be established as a stand-alone WHO manual instead of being integrated into
the current WHO Recommendations to allow for more rapid and flexible
adaptations to real-world changes than would otherwise be possible in the
context of a full revision of the latter.
The Committee recommended that the WHO manual be adopted and
annexed to its report (Annex 6). Additionally, the Committee also recommended
that part B of the current WHO Recommendations for the preparation,
characterization and establishment of international and other biological reference
standards be revised so that it referred to the new guidance.
Recommendations for the preparation, characterization and establishment of international and other
8
biological reference standards (revised 2004). In: WHO Expert Committee on Biological Standardization:
fifty-fifth report. Geneva: World Health Organization; 2006: Annex 2 (WHO Technical Report Series, No. 932;
http://www.who.int/immunization_standards/vaccine_reference_preparations/TRS932Annex%202_
Inter%20_biol%20ef%20standards%20rev2004.pdf?ua=1, accessed 23 February 2017).
41
WHO Expert Committee on Biological Standardization Sixty-seventh report
The Committee asked how often IVD manufacturers are inspected and
were informed that inspections take place following every new application.
However, if a production line of interest had been inspected recently, an
inspection lasting 1 day (instead of 3 days) may be sufficient, depending on the
risk-based decision made. Further discussion included the observation that for
the EUAL procedure, post-marketing follow-up is currently not funded making
it difficult to perform, and the clarification that quality control laboratories can
apply to perform appropriate evaluations should they wish.
The Committee agreed that the relevant Technical Specification
Series documents being produced by the WHO PQ Team in the area of IVD
prequalification should be sent to the Committee for review and endorsement.
42
International Recommendations, Guidelines and other matters
Guidelines for the safe production and quality control of inactivated poliomyelitis vaccine manufactured
9
from wild polioviruses (Addendum, 2003, to the Recommendations for the production and quality
control of poliomyelitis vaccine (inactivated)). In: WHO Expert Committee on Biological Standardization:
fifty-third report. Geneva: World Health Organization; 2004: Annex 2 (WHO Technical Report Series,
No. 926; http://www.who.int/biologicals/publications/trs/areas/vaccines/polio/Annex%202%20(65-89)
TRS926Polio2003.pdf?ua=1, accessed 2 February 2016).
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WHO Expert Committee on Biological Standardization Sixty-seventh report
There was also agreement that the proposed scope of the revised
document should encompass: (a) containment of the manufacturing and quality
control facilities for inactivated poliomyelitis vaccine (IPV) derived from both
Sabin and wild-type poliovirus; and (b) a risk assessment of new safer strains of
poliovirus to be used in manufacturing or quality control testing. The routine
production of OPV using Sabin vaccine strains would be outside the scope of
the document because as long as it is being used for routine immunization
it does not have to be contained. When it is not in routine use it will not be
made, leaving only the problem of manufacturing a post-eradication OPV for
emergency response – assuming that the ability to make OPV is retained. It
should also be possible to store contained poliovirus, such as monovalent OPV,
outside GAPIII-compliant facilities which would greatly simplify future work
for manufacturers and other stakeholders. Several other issues had yet to be
resolved, such as the need for routine showering upon exit from the containment
area as required by GAPIII.
The Committee noted with interest the information provided and,
following discussion and clarification of a number of points, expressed its
support for the developments to date and looked forward to hearing of further
progress at its next meeting.
by the Committee in 2007 and provide NRAs and vaccine manufacturers
with: (a) guidance on regulatory pathways for approving pandemic influenza
vaccines; (b) regulatory considerations to be taken into account in evaluating
the quality, safety and efficacy of candidate pandemic influenza vaccines; and
(c) guidance on effective post-marketing surveillance of pandemic influenza
vaccines. However, these Guidelines apply mainly to countries in which
influenza vaccine production takes place and where pandemic influenza
vaccines are likely to be given market authorization first in the event of a
pandemic. Consultation with stakeholders following the 2009 H1N1 influenza
pandemic identified regulatory delays due to a lack of regulatory preparedness
as a significant factor in delaying or preventing the deployment of pandemic
vaccine in non-vaccine-producing countries. This was especially the case for
44
International Recommendations, Guidelines and other matters
WHO Recommendations for the production and control of influenza vaccine (inactivated). In: WHO Expert
10
Committee on Biological Standardization: fifty-fourth report. Geneva: World Health Organization; 2005:
Annex 3 (WHO Technical Report Series, No. 927; http://apps.who.int/iris/bitstream/10665/43094/1/WHO_
TRS_927_eng.pdf, accessed 23 February 2017).
46
International Recommendations, Guidelines and other matters
indicating that, on the basis of current evidence, the use of IIV in pregnant
women is not contraindicated. It was expected that this would in turn facilitate
maternal immunization programmes by raising awareness of the convergence
of regulatory positions on this matter. The Committee was informed that the
proposed document (WHO/BS/2016.2280) had been the subject of extensive
international public consultation with regulators, manufacturers, academia and
vaccine users, which had resulted in editorial changes to improve clarity.
Following review of the document, the Committee considered it to
be a suitable explanatory document without further revision, and therefore
recommended that the proposed addendum be adopted and annexed to its
report (Annex 8).
Guidelines on clinical evaluation of vaccines: regulatory expectations. In: WHO Expert Committee on
11
Biological Standardization: fifty-second report. Geneva: World Health Organization; 2004: Annex 1 (WHO
Technical Report Series, No. 924; http://www.who.int/biologicals/publications/trs/areas/vaccines/clinical_
evaluation/035-101.pdf?ua=1, accessed 23 February 2017).
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WHO Expert Committee on Biological Standardization Sixty-seventh report
During the course of the revision process for the WHO Guidelines on clinical
evaluation of vaccines: regulatory expectations (see section 3.5.4 above) it
became clear that one subject area that was not covered by the original 2001
document, and which might be considered for addition during revision, was
human challenge trials. Human challenge trials are studies in which immunized
and non-immunized volunteers are intentionally challenged with an infectious
disease-causing organism to see whether immunization affords any protection
against the challenge strain. The challenge organism may be close to wild-type
and pathogenic (but adapted and/or attenuated from wild-type, with possibly
less or no pathogenicity) or genetically modified in some way. During subsequent
public consultations, it was agreed that since human challenge trials are not
required for licensing, any guidance on this matter should be developed as a
48
International Recommendations, Guidelines and other matters
vaccines. The first drafts of the Guidelines were thus concerned with an epidemic
situation with a focus on accelerating product development and availability
during a public health emergency. The end of the large-scale EVD outbreak in
Africa and the post-emergency epidemiological situation made the assessment of
Ebola vaccine efficacy challenging due to the now sporadic nature of the disease.
The Committee was informed that document WHO/BS/2016.2279
provides scientific and regulatory guidance for NRAs and vaccine manufacturers
on the quality, and nonclinical and clinical aspects of Ebola vaccines relevant to
marketing authorization applications. The Guidelines particularly apply to Ebola
vaccines based on viral vectors as these are the vaccines currently at the most
advanced stages of development and for which no specific WHO guidance is
available. For the first time in any WHO Guidelines of this type, opportunities
to accelerate product development and availability during a public health
emergency are discussed.
Although the Committee recognized that ongoing clinical studies may
generate more data on vaccine performance, it agreed that any new data were
unlikely to significantly alter the technical guidance given. Furthermore, the
early availability of such WHO Guidelines in the public domain would aid the
evaluation of applications for clinical trials and for the licensure of Ebola vaccines
in the near future. The Guidelines may also be of benefit in the evaluation of
other viral vectored vaccines and could help guide future activities in outbreak
situations, especially those which constituted a Public Health Emergency of
International Concern. At the same time, specific outbreaks have unique features
and there would always be a need for flexibility of response.
The Committee reviewed the draft document WHO/BS/2016.2279
and after extensive discussion agreed that the guidance given on multivalent
Ebola vaccines and on the clinical evaluation of candidate vaccines using newer
clinical trial designs should be expanded. A revised document will therefore be
WHO Technical Report Series, No. 1004, 2017
50
4. International reference materials –
biotherapeutics other than blood products
All reference materials established at the meeting are listed in Annex 11.
51
WHO Expert Committee on Biological Standardization Sixty-seventh report
products to ensure their clinical safety and efficacy. The intended users of such
standards would be manufacturers and regulatory authorities. Ideally, candidate
materials from both originator and SBP manufacturers would be included in
a collaborative study. The proposed NIBSC plan is to initiate activities with
bevacizumab or ranibizumab (depending on the availability of materials) and
to follow on with other molecules as available. Pilot lyophilizations would be
conducted to determine a suitable formulation for the molecule of interest.
Following definitive fills, the different candidate preparations would then be
included in an international collaborative study. The range of assays likely to be
used would include binding assays and bioassays involving both primary and
engineered cell lines.
The Committee regarded this as a longer term project with somewhat
unclear timelines, and it was envisaged that securing candidate materials for the
52
International Recommendations, Guidelines and other matters
the potency testing and lot release of these products. To date, developers,
manufacturers and regulators have relied solely on the use of in-house qualified
reference standards and the reference clinical product during development
and manufacturing, and no higher-order of mAb standard for bioassays is
currently available.
Given that mAbs are the fastest-growing class of biotherapeutic product,
and with increasing numbers of SBPs now in development, there is a widely
recognized need for the global standardization of biotechnology products in this
field. Preliminary data from ongoing collaborative studies for the development
of WHO international standards and reference reagents for mAbs such as
rituximab are encouraging, and highlight the value of such reagents in assessing,
and ensuring the consistency of, bioassay performance by different stakeholders.
The proposal to develop WHO international standards or reference reagents for
54
International Recommendations, Guidelines and other matters
use in the calibration, evaluation and validation of bioassays for measuring the
biological activity of mAbs to the ErbB/HER receptor family is aligned with
these recognized needs.
NIBSC therefore proposed an international multicentre collaborative
study to evaluate the suitability of candidate preparations to act as biological
standards/reference reagents in one or more relevant direct or indirect functional
assays. Depending on the specific mAb under study, these could include
bioassays for measuring cell binding, inhibition of receptor signalling, inhibition
of proliferation of target cells and/or antibody-dependent cytotoxicity.
Candidate reference materials would now be sought from an innovator
or SBP manufacturer. As this may be challenging, the first study to be carried
out will be determined by the ability to source suitable candidate material.
Other preparations may also be tested in parallel for comparison. It was
expected that an international collaborative study would be likely to take place
in 2017 and 2018.
The Committee agreed that there was a need for the standardization of
biotechnology products globally and endorsed the proposal (WHO/BS/2016.2296
Rev.1) to develop international standards and/or reference reagents for use in
this area. It noted that, ideally, the relevant reference materials should become
available as SBPs were being developed and licensed, and that there was now a
need for a catch-up programme of work.
55
5. International reference materials – blood
products and related substances
All reference materials established at the meeting are listed in Annex 11.
reference reagent (NIBSC code 15/140) for use as the First WHO Reference
Reagent for batroxobin.
A total of 17 laboratories returned data, with each asked to perform four
independent assays. A range of assay methods were encouraged and suitable
protocols provided. Potency estimates for ancrod were made relative to the
current standard using parallel-line analysis and an unweighted geometric mean
potency of 53.9 IU/ampoule obtained, consistent with the expected potency of
55 IU/ampoule. Greater variability was observed in the batroxobin values, with
additional data from assays independent of the study confirming an apparent
10% higher potency when using plasma as the substrate compared to fibrinogen,
thus highlighting a small but significant discrepancy between the proposed
and current materials – potentially due to degradation of the current standard.
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International reference materials – blood products and related substances
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WHO Expert Committee on Biological Standardization Sixty-seventh report
countries taking part in the assignment of FXI:Ag values. In all cases, the use
of four independent assays was requested, each with four or more dilutions in
duplicate. All data were analysed by parallel-line assay, for which only assays
valid for linearity were used (p > 0.01). Parallelism was assessed as the ratio of
standard and test slopes. Potency estimates were calculated using the unweighted
geometric mean.
Excellent inter-laboratory agreement was observed for the assignment
of FXI:C value (geometric coefficient of variation (GCV) = 1.8%) and no
discrepancy was observed between the IU and the plasma unit (0.71 versus
0.72 IU/ampoule). For FXI:Ag value assignment, there was also good
agreement between laboratories, albeit with wider variation due to differences
between plasma pools. A small method bias was also seen, which is unlikely to
be significant.
During discussion, the Committee noted that although enzyme activity
had remained stable during stability studies, the antigenicity had dropped.
Although this was the opposite of what would be expected, this may have
resulted from the different reference products used or to the higher variability of
the antigenicity test results. It was also commented that in future such validation
studies of international reference preparations should involve laboratories from
a wider selection of WHO regions.
The Committee considered the report of the study (WHO/BS/2016.2281)
and recommended that the candidate material 15/180 (6000 ampoules) be
established as the Second WHO International Standard for blood coagulation
factor XI (plasma, human) with an FXI:C value of 0.71 IU/ampoule, and an
FXI:Ag value of 0.78 IU/ampoule.
four freeze-dried control plasmas were used. To avoid bias in the testing system,
the order of testing was different from day to day. The mean ISIs obtained were
1.11 for candidate 14/001 (inter-laboratory coefficient of variation = 5.7%) and
1.21 for candidate 15/001 (inter-laboratory coefficient of variation = 4.6%).
Accelerated degradation studies were also performed to assess the stability of the
freeze-dried candidate materials. There was no significant change in PT following
storage of the candidate materials at 5 °C for 56 days. After storage at elevated
temperatures (31–42 °C) there was a slight but significant change in PT. Stability
after reconstitution was assessed at room temperature, over a time interval of
0.5–4 hours. Between 1 and 4 hours after reconstitution there was no change
in PT for candidate material 14/001 and a slight change (−1.5%) for candidate
material 15/001. Should these materials be established for use as international
standards, it was proposed that the provisional codings used above be replaced
by rTF/16 and RBT/16 respectively.
The results revealed significant variation in ISI results between
laboratories, indicating that further standardization of the manual tilt tube
technique is needed. It was intended that a document would be prepared
that provided full details of the technique for approval by the Scientific and
Standardization Committee of the International Society on Thrombosis and
Haemostasis (SSC/ISTH). The document was then to be submitted as an
approved reference method to the Joint Committee for Traceability in Laboratory
Medicine of the Bureau International des Poids et Mesures.
The Committee noted that an automated method would be a good way
of standardizing results as the manual tilt tube technique was apparently prone
to variability. Despite a number of potential methodological challenges, a strong
recommendation was made by the Committee to include automated methods
in future validation studies of reference preparations for thromboplastins.
Furthermore, given the observed instability of the candidate materials at
temperatures > 30 °C, it was proposed that written advice to ship them with
cooling packs to maintain temperatures at or below controlled room temperature
be added to the product Instructions for Use.
The Committee considered the report of the study (WHO/BS/2016.2294)
and recommended that the candidate material 14/001 (to be re-coded as rTF/16)
be established as the Fifth WHO International Standard for thromboplastin
(recombinant, human, plain) with an assigned value of 1.11 IU/ml.
5.2.3 Proposed assignment of factor XIII-B subunit (total and free) values
to the First WHO International Standard for factor XIII plasma
The First WHO International Standard for factor XIII plasma is currently used to
measure the potency (functional activity and antigen value) of blood coagulating
factor XIII (FXIII) in patient plasma during the diagnosis of FXIII deficiencies,
and also to evaluate FXIII therapeutic concentrates. FXIII circulates in plasma
as a heterotetramer of two A and two B subunits (FXIII-A2B2) in a 1:1 complex.
The active A subunit functions by cross-linking fibrin and stabilizing the fibrin
structure. The B subunit is a carrier protein without activity. However, FXIII-B is
in excess over FXIII-A with around 50% of total FXIII-B existing in complex with
FXIII-A and around 50% in free form. The half-life of FXIII-A depends upon
the amount of available FXIII-B. Congenital and acquired FXIII deficiencies are
severe bleeding disorders, and FXIII-B measurements are crucial in correctly
diagnosing and characterizing the type of FXIII deficiency. Furthermore, therapy
for FXIII-A deficiency with recombinant FXIII-A relies on available FXIII-B.
Free FXIII-B may also have other so far unknown functions.
It was proposed that an international multicentre study involving
manufacturers, clinical and research laboratories, and regulatory authorities be
conducted to calibrate both the total and free levels of the FXIII-B subunit in
the First WHO International Standard for factor XIII plasma relative to levels in
locally collected and pooled normal plasma. Calibration will be performed by
specific ELISAs with value assignment for the B subunit made in IUs. In order to
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WHO Expert Committee on Biological Standardization Sixty-seventh report
determine both total and free FXIII-B levels, mAb to the free FXIII-B subunit is
required and has recently been developed – although availability was restricted
to a single supplier. These antibodies have been made available free of charge for
the proposed value-assignment study but have also been commercialized and
will thereafter be available for purchase. This proposal could also be viewed as
part of the standardization of a companion diagnostic for informing the use of
a recombinant FXIII-A product (Novo XIII) which should not be administered
without assessing the patient’s FXIII-B subunit levels.
The Committee noted that approximately 150–200 ampoules are
currently dispatched each year and that this level of demand was likely to increase
with the new proposed value assignment. The Committee also emphasized that
FXIII deficiency can also be treated with plasma. The Committee then endorsed
the proposal (WHO/BS/2016.2297) to assign additional analyte potencies to the
current First WHO International Standard for factor XIII plasma.
some antibodies and do not allow for the measurement of TAFIa. Assays based
on functional activity measurement are available – either direct-activity-based
assays (complicated in plasma by carboxypeptidase N activity) or indirect
measurements that require the quantitative activation of TAFI. Both these assay
types present challenges, including lower specificity when using direct assays.
To obtain reliable results from these different assays, standardized
methods are required, and a common reference material for TAFI is now needed
to harmonize global measurements. Such a material would be intended for use
by manufacturers of commercial kits and by academic and clinical laboratories
investigating TAFI as a disease marker. It was therefore proposed that a study
be conducted involving all available antigen and activity assay methods. It was
recognized that such an approach may yield highly variable results, particularly
as ELISA-based assays measure concentration and not activity, and that the
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International reference materials – blood products and related substances
64
6. International reference materials –
cellular and gene therapies
All reference materials established at the meeting are listed in Annex 11.
66
7. International reference materials – in vitro diagnostics
All reference materials established at the meeting are listed in Annex 11.
for in vitro transcripts only occurred when expressed relative to other in vitro
transcripts, with no harmonization observed when the candidate material was
expressed relative to the universal in vitro transcript.
Discussion then took place of possible explanations for an observed
variability between two laboratories in the degree of harmonization of results
against the candidate material when material of African lineage was assayed.
The Committee considered that the discrepant results might have been due
to the different methods of virus inactivation used or to the matrix used for
lyophilization, rather than the African origin of the isolates per se. In relation to
the results observed for in vitro transcripts, it was noted that armored RNA was
not available at the time of the study, but may provide a solution for improving
harmonization in this regard. It was agreed that the continued fitness for
purpose of the candidate material should be monitored against new isolates as
they occur and that further data should be generated using NAT-based assays
of African isolates.
The Committee considered the report of the study (WHO/BS/2016.2286)
and recommended that the candidate material 11468/16 be established as the
First WHO International Standard for Zika virus RNA for NAT-based assays
with an assigned content of 50 000 000 IU/ml.
7.1.2 First WHO Reference Panel for Ebola virus VP40 antigen
Although the EBOV outbreak in West Africa has now ended, WHO preparedness
activities are continuing to ensure that all countries are operationally ready to
effectively and safely detect, investigate and report potential EVD cases, and to
mount effective responses. There is thus a need for the development of point-
of-care rapid tests for the specific and accurate EVD diagnosis, which do not
require complex laboratory equipment or highly trained staff.
An international collaborative study was therefore conducted to evaluate
WHO Technical Report Series, No. 1004, 2017
were used, which included point-of-care tests, research use only tests and tests
approved for use in the WHO EUAL procedure.
Eight laboratories from four countries each received nine blinded study
samples and were asked to reconstitute these and test them without dilution.
Each sample was assessed in three different runs and the results reported as either
positive or negative. In general, results were consistent across the different VP
assays with few exceptions. Assays of nucleoprotein and glycoprotein contained
in the preferred candidate material almost invariably produced negative results
for samples demonstrated as positive for VP40. As only limited stability data
were available, it was acknowledged that additional studies would be required
before any long-term stability predictions could be made. It was also noted that
the moisture content in the novel vials was higher than is usually observed in
ampoules. It was proposed that the preferred candidate medium- and low-titre
VP40 samples plus a negative sample should be used to form a reference panel
for use by laboratories in the qualitative assessment of assay performance in
detecting VP40, with the potential limitations of the materials to be made clear
to end users. The panel would not be suitable for assessing assays used to detect
EBOV antigens other than VP40, and further work would be needed to produce
reference materials for glycoprotein and nucleoprotein assays.
The Committee discussed the suitability of VP40 expressed in bacterial
cells as a reference material for assays for point-of-care use. It was recommended
that this aspect should be highlighted in the Instructions for Use of the proposed
reference panel, and that the future inclusion of reference reagents for VP40
expressed in mammalian cells should be considered.
The Committee considered the report of the study (WHO/BS/2016.2302)
and recommended that the candidate materials be established as the First WHO
Reference Panel for Ebola virus VP40 antigen with the provision that it is clearly
indicated that the material was only suitable for VP40 assays and that some
assays might fail to detect a non-envelope-associated antigen.
for recent DENV infection, and a proposal by the United States Food and Drug
Administration to prepare standards for use in this area had been endorsed by
the Committee in 2009.
Four candidate materials (one for each of the serotypes 1–4) had been
prepared, heat-inactivated and diluted into a base matrix at a concentration
of approximately 6 log10 PCR-detectable units/ml. Both liquid frozen and
lyophilized materials were prepared for each serotype (2000 and 8000 vials
respectively). The materials were then evaluated in an international collaborative
study during which 21 laboratories in 15 countries returned data. A wide range
of extraction and amplification methods were used, with most assays using a
different target region for amplification. Observed variations in intra-laboratory
estimates indicated a lack of reproducibility even within the same test, and
considerable variability was also observed in data sets produced by two of the
four laboratories that had used quantitative methods. However, despite these
issues, all assays were able to detect the viral RNA of each serotype, with negative
samples also correctly identified. Proposed potency estimates were assigned
to the proposed reference materials for each serotype with values reported as
NAT-detectable units (NDUs) and a 95% confidence interval established for each
data value.
The Committee agreed that there was a need for these materials. During
discussion, the subject of value assignment was raised, and in particular the
potential for confusion which may arise from the use of the term “NDU”. There
was also much discussion as to whether the materials should be established
as an international reference panel, as four separate reference reagents or as
international standards for each serotype, with a case for each option being
made. Following further deliberation, the Committee concluded that the
materials should be established as four separate WHO reference reagents, each
with an assigned value (“unit”) without a cited confidence interval. It was noted
that these reference materials could be further developed as DENV serotype-
WHO Technical Report Series, No. 1004, 2017
The Committee noted that one laboratory had reported only limited
commutability and suggested that further work should be undertaken with
the assay manufacturer to understand the reason for this. The Committee also
questioned whether manufacturers use recombinant materials for their kit
controls and were informed that end users are not provided with this information.
The Committee commended the commutability work undertaken for this
study but noted that problems in sourcing suitable materials for commutability
assessments in this area should not be underestimated.
The Committee considered the report of the study (WHO/BS/2016.2292)
and recommended that the candidate material 83/573 be established as the
Fourth WHO International Standard for prolactin (pituitary, human) with an
assigned value of 67 mIU/ampoule.
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International reference materials – in vitro diagnostics
obtained. This was unexpected based on the initial in-house validation of the
materials used. Additional data also need to be generated on the stability of
the candidate reference preparation. Once all analysis is completed, a report will
be prepared.
The Committee suggested that in the final report potency should be
assigned relative to the interim standard. It was then proposed that, as the
finalization of the report would occur well in advance of the 2017 meeting
of the Committee, the material could be made available before its formal
establishment as an international standard. Following some discussion around
this point, it was agreed that the material should remain labelled as a “candidate
standard” with a value assigned in units and not IUs – which could be changed
if the Committee formally established the material in 2017. Accordingly, the
Committee recommended that, following the completed analysis, the candidate
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International reference materials – in vitro diagnostics
7.2.2 Proposed First WHO International Standard for Zika virus antibodies
(immunoglobulin G and immunoglobulin M) (human)
The accurate diagnosis of ZIKV infection, particularly in pregnant women, is
a crucial step in making appropriate health-care decisions. However, the virus
has a short period of detection in plasma, and PCR-based tests of plasma are
thus only useful during this period (approximately 9 days following the onset
of symptoms). After a negative PCR result, diagnosis is made on the basis of
serological testing that measures antibody responses to the virus. Current
serological tests are prone to high cross-reactivity against other flaviviruses
(particularly DENVs) spread by the same mosquito vector. To ensure accurate
diagnosis, the standardization of tests is required to improve both their sensitivity
and specificity. Both immunoglobulin G and immunoglobulin M are currently
measured to determine prior exposure, where immunoglobulin M would indicate
a recent infection. To support the WHO response to the ZVD outbreak NIBSC
had initiated development of an international standard for use in the calibration
and control of ZIKV antibody assays.
The intended use of such a material would be the calibration
of ELISAs and neutralization assays used to measure ZIKV antibody
levels (immunoglobulin G and immunoglobulin M) in human serum. The
recommendation to use the candidate material for this purpose will be subject
to the satisfactory demonstration of commutability. Primary users will be public
health and other clinical laboratories, kit manufacturers and ZIKV vaccine
manufacturers (for clinical trials).
The Committee expressed concern over reported delays and problems
encountered in receiving positive plasma donations from countries with infected
individuals. It was noted that difficulties were also encountered in sourcing ZIKV
antibody-positive plasma that was negative for antibodies to other flaviviruses.
The Committee endorsed the proposal (WHO/BS/2016.2298 Rev.1) to develop a
First WHO International Standard for Zika virus antibodies (immunoglobulin G
and immunoglobulin M) (human).
Asia and the Indian subcontinent and, since 2013, has spread to the Americas,
particularly central and southern areas. Small outbreaks have also occurred
recently in Europe. The diagnosis of CHIKV infection requires a variety of
tests, including the detection of immunoglobulin M and immunoglobulin G
antibodies. The co-circulation of CHIKV with DENV and ZIKV frequently
occurs and infections caused by these viruses share common signs and symptoms
in infected patients.
The accurate diagnosis and discrimination of CHIKV infection from
other virus infections is thus vital for patient care. Analysis of immunoglobulin M
antibodies is particularly useful for the confirmation of acute infection. Anti-
CHIKV immunoglobulin G may also be detectable during acute infection, but
is also a marker of past CHIKV infection and of seroprevalence. Anti-CHIKV
assays vary in their performance, and Alphavirus serological cross-reactivity is
known to exist with members of the Semliki Forest serocomplex.
A collaborative study was therefore proposed to evaluate sera from
CHIKV-infected patients (and potentially from blood donors) for their
suitability for use as an international standard. The samples would include both
immunoglobulin G and immunoglobulin M reactive sera to distinguish between
recent and past infections.
The Committee was informed that sera would be sourced through national
and international collaborations, with the proposed project complementing
a previously endorsed and ongoing CHIKV RNA project. The Committee
endorsed the proposal (WHO/BS/2016.2298 Rev.1) to develop a First WHO
International Standard for chikungunya virus antibodies (immunoglobulin G
and immunoglobulin M) (human).
batch of tissue-culture-grown virus (HIV-2 CAM-2) as was used for the current
international standard. Approximately 500 vials of lyophilized material would
be produced.
The Committee endorsed the proposal (WHO/BS/2016.2298 Rev.1) to
develop a Second WHO International Standard for human immunodeficiency
virus type 2 RNA for NAT-based assays.
member at a 10–20% dilution ratio was suggested. There was also some discussion
concerning the decision not to include lyophilized cells, with the exclusive use of
extracted DNA viewed as a positive step in assay standardization. It was clarified
that the cell line used in the development of the BRAF material had been derived
from an Epstein-Barr virus-positive cell line and it would not be appropriate to
supply laboratories with a class-2 pathogen in addition to the genetic material,
with variability in DNA extraction methods additionally compromising the
reference value of the DNA standard.
The Committee endorsed the proposal (WHO/BS/2016.2298 Rev.1) to
develop a First WHO Reference Panel for the BRAF V600E gene mutation.
(NIST) has prepared a genomic standard for HER2 this is for genomic DNA
measurement only. Envisaged users include manufacturers (for the calibration
of diagnostic kits) and clinical and reference laboratories (for the calibration
of secondary standards used in multiple routine diagnostic assays for ErbB2
characterization). The project is predicted to progress such that the results for
the panel to be established would be submitted to the Committee in 2020.
The Committee endorsed the proposal (WHO/BS/2016.2298 Rev.1)
to develop a First WHO Reference Panel for ErbB2 copy number and mRNA
expression.
80
8. International reference materials –
vaccines and related substances
All reference materials established at the meeting are listed in Annex 11.
(around 3 per 1000 live births) these vaccines were likely to be licensed on the
basis of surrogates of protection (immunoglobulin G level and assessment of
functional antibody by opsonophagocytosis assay) rather than through very
large clinical protection studies. There is, therefore, an urgent need to establish
standardized assays for the quality control of such vaccines and to measure the
concentration and functional activity of antibodies against GBS in the sera of
immunized individuals.
The development of several standards and reference reagents
(polysaccharide standards and human reference serum) is required. The
polysaccharide standards would be used to calibrate internal controls in various
physicochemical and immune assays used in the quality control of GBS vaccines
(identity, total and free polysaccharide) and as antigens to measure antibody
responses. Human reference sera will be used as standards in assays for the
quantification of antibody response and for measurement of functional activity
in clinical trials materials. It was expected that vaccine manufacturers would
donate the polysaccharides and that human serum reference materials would be
obtained from volunteers immunized with the conjugate vaccine. It was proposed
that monovalent sera already available at NIBSC should be used to calibrate
the human reference serum. These monovalent sera have been calibrated by
radio-immunoassays for antibody concentration and have been looked upon
as gold standards in various studies to establish correlates of protection, and by
manufacturers in evaluating immune responses during clinical trials.
It was expected that the availability of relevant international standards
would expedite the development and licensing of GBS vaccines for immunizing
pregnant women in order to prevent GBS in newborns, as well as facilitate
ongoing quality control testing should such vaccines be licensed. Envisaged
users include vaccine and diagnostic kit manufacturers, public and national
health authorities, academic researchers and NCLs.
Following discussion and clarifications, the Committee endorsed the
WHO Technical Report Series, No. 1004, 2017
HPV6 and HPV11 and against high-risk types HPV31, HPV33, HPV45, HPV52
and HPV58 would provide the full complement of international standards for
use in standardizing HPV serology assays. The users of such reference materials
would include vaccine developers and manufacturers, epidemiologists, research
laboratories, public health laboratories and developers of assay kits.
For each anti-HPV type, donations will be obtained from at least two
individuals naturally infected with the HPV type of interest. Sera would preferably
be reactive with only one genital HPV type, with donation suitability assessed by
testing for antibodies against multiple genital HPV types.
The International HPV Reference Center, Karolinska Institute will be
involved in enrolling and selecting suitable donors, with candidate sera having
also been obtained from collaborators at the National Institutes for Food and
Drug Control, China. NIBSC will undertake filling and freeze-drying operations
for each candidate standard according to standard operating procedures. The
freeze-dried candidates will then be assessed in an international collaborative
study involving 10–15 laboratories using a range of assays. Assay data will be
analysed at NIBSC using standard statistical techniques. Stability studies on the
candidate standards will also be carried out in the usual way. It was expected
that sourcing suitable samples from naturally infected individuals to produce
the seven international standards may be the rate-limiting step for this project.
Following discussion and clarifications, the Committee endorsed the
proposal (WHO/BS/2016.2295) to develop WHO international standards for
antibodies against human papillomavirus types 6, 11, 31, 33, 45, 52 and 58.
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Annex 1
WHO Recommendations, Guidelines and other documents
related to the manufacture, quality control and evaluation
of biological substances used in medicine
WHO Recommendations, Guidelines and other documents are intended to
provide guidance to those responsible for the production of biological substances
as well as to others who may have to decide upon appropriate methods of
assay and control to ensure that products are safe, reliable and potent. WHO
Recommendations (previously called Requirements) and Guidelines are scientific
and advisory in nature but may be adopted by a national regulatory authority
(NRA) as national requirements or used as the basis of such requirements.
Recommendations concerned with biological substances used in
medicine are formulated by international groups of experts and are published
in the WHO Technical Report Series 1 as listed below. A historical list of
Requirements and other sets of Recommendations is available on request from
the World Health Organization, 20 avenue Appia, 1211 Geneva 27, Switzerland.
Reports of the WHO Expert Committee on Biological Standardization
published in the WHO Technical Report Series can be purchased from:
WHO Press
World Health Organization
20 avenue Appia
1211 Geneva 27
Switzerland
Telephone: + 41 22 791 3246
Fax: +41 22 791 4857
Email: [email protected]
Website: www.who.int/bookorders
Individual Recommendations and Guidelines may be obtained free of
charge as offprints by writing to:
Technologies Standards and Norms
Department of Essential Medicines and Health Products
World Health Organization
20 avenue Appia
1211 Geneva 27
Switzerland
87
WHO Expert Committee on Biological Standardization Sixty-seventh report
Blood plasma products (human): viral Adopted 2001, TRS 924 (2004)
inactivation and removal procedures
Blood regulatory systems, assessment criteria Adopted 2011, TRS 979 (2013)
for national
Cholera vaccines (inactivated, oral) Adopted 2001, TRS 924 (2004)
Dengue tetravalent vaccines (live, attenuated) Revised 2011, TRS 979 (2013)
Diphtheria, tetanus, pertussis (whole cell), and Revised 2012, TRS 980 (2014)
combined (DTwP) vaccines
Diphtheria vaccines (adsorbed) Revised 2012, TRS 980 (2014)
DNA vaccines: assuring quality and nonclinical Revised 2005, TRS 941 (2007)
safety
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Annex 1
Rabies vaccines for human use (inactivated) Revised 2005, TRS 941 (2007)
produced in cell substrates and embryonated
eggs
Reference materials, secondary: for NAT-based Adopted 2016, TRS 1004 (2017)
and antigen assays: calibration against WHO
International Standards
Regulation and licensing of biological products Adopted 1994, TRS 858 (1995)
in countries with newly developing regulatory
authorities
Regulatory risk evaluation on finding an Adopted 2014, TRS 993 (2015)
adventitious agent in a marketed vaccine:
scientific principles
90
Annex 1
92
Annex 2
Guidelines on evaluation of monoclonal antibodies as
similar biotherapeutic products (SBPs)
1. Introduction 97
2. Purpose and scope 98
3. Terminology 99
4. Special considerations for characterization and quality assessment 101
4.1 Strategy for assessment of mAb biological activity 101
4.2 Considerations for selection of the expression system 103
4.3 International standards for biological assays used in the characterization 104
5. Special considerations for nonclinical evaluation 104
5.1 In vitro studies 104
5.2 In vivo studies 106
6. Special considerations for clinical evaluation 109
6.1 Pharmacokinetics studies 109
6.2 Pharmacodynamics studies 112
6.3 Comparative clinical efficacy study 113
6.4 Indication extrapolation 122
6.5 Pharmacovigilance and post-approval consideration 123
Authors and acknowledgements 123
References 125
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WHO Expert Committee on Biological Standardization Sixty-seventh report
94
Annex 2
Abbreviations
ACR20 American College of Rheumatology 20% improvement criteria
ADA anti-drug antibody
ADCC antibody-dependent cellular cytotoxicity
ADCP antibody-dependent cellular phagocytosis
AUC area under the curve
CDC complement-dependent cytotoxicity
CHO Chinese hamster ovary
CLB competitive ligand-binding (assay)
CR complete response
CRP C-reactive protein
DAS28 disease activity score in 28 joints
DCVMN Developing Countries Vaccine Manufacturers Network
EGFR epidermal growth factor receptor
ESR erythrocyte sedimentation rate
IFPMA International Federation of Pharmaceutical Manufacturers
& Associations
IgE immunoglobulin E
IgG immunoglobulin G
IGPA International Generic Pharmaceutical Alliance
mAb monoclonal antibody
MOA mechanism of action
NRA national regulatory authority
ORR overall response rate
pCR pathological complete response
PD pharmacodynamics
PK pharmacokinetics
RBP reference biotherapeutic product
rDNA recombinant deoxyribonucleic acid
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WHO Expert Committee on Biological Standardization Sixty-seventh report
96
Annex 2
1. Introduction
Monoclonal antibodies (mAbs) are a major class of recombinant deoxyribonucleic
acid (rDNA) technology-derived biotherapeutic products that have achieved
outstanding success in treating many life-threatening and chronic diseases. Some
of these targeted therapy products are ranked in the top-10 lists of annual global
pharmaceutical revenue sources. As patents and data-protection measures on
mAb products have expired, or are nearing expiry, considerable attention has
turned towards producing similar biotherapeutic products (SBPs, also termed
“biosimilars”) based upon the approved mAb innovator products, with a view
to making more affordable products that could improve global access to these
so‑called blockbusters.
Therapeutic mAbs are preparations of an immunoglobulin or a fragment
of an immunoglobulin with specificity for a target ligand and are derived from
a single clone of cells. Each full-length molecule of a mAb consists of two heavy
and two light polypeptide chains which are linked by disulfide bonds. MAbs
have several possible functional domains within a single molecule. The defined
specificity of a mAb is based on the binding region for an antigen that is located
in the antigen-binding fragment (Fab) region. For full-length mAbs, their
crystallizable fragment (Fc) region has the ability to bind to specific receptors,
potentially leading to immune effector functions such as antibody-dependent
cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC)
and antibody-dependent cellular phagocytosis (ADCP). Full-length mAbs
are glycoproteins with glycosylation sites in the Fc region of the heavy chains,
with further possible glycosylation sites depending on the type of molecule.
Therefore, mAbs are highly complex biological macromolecules with size and
charge variants, various post-translational modifications including different
glycosylation patterns and N- and C-terminal heterogeneity, long half-lives and
the potential to induce immunogenicity. Each individual mAb may therefore
present a unique profile, which should be taken into consideration during the
evaluation of such products as SBPs.
The WHO Guidelines on evaluation of similar biotherapeutic products
(SBPs) were adopted by the WHO Expert Committee on Biological
Standardization in 2009 (1). This document set out the scientific principles,
including the stepwise approach, which should be applied for the demonstration
of similarity between an SBP and the reference biotherapeutic product (RBP).
High similarity at the quality level is regarded as a prerequisite for enabling the
use of a tailored nonclinical and clinical programme for licensure. The goal of
the clinical comparability exercise is to confirm the similarity established at
previous stages of development and to demonstrate that there are no clinically
meaningful differences between the SBP and the RBP – and not to re-establish
safety and efficacy, as this has been done already for the RBP. The decision on
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WHO Expert Committee on Biological Standardization Sixty-seventh report
licensure of the SBP should be based upon evaluation of the totality of evidence
from quality, nonclinical and clinical parameters. It should be noted that clinical
studies cannot be used to resolve substantial differences in physicochemical
characteristics and biological activity between the RBP and the SBP. If substantial
differences in quality attributes are present, a stand-alone licensing approach
may be considered.
The set of globally acceptable key principles outlined above for the
regulatory evaluation and licensing of SBPs has served well as a basis for setting
national requirements for SBPs. However, because of the structural complexity
and heterogeneity of mAbs, their quality attributes can vary from product
to product. Furthermore, one mAb product may have multiple indications.
Therefore, biosimilar comparability studies between a candidate biosimilar mAb
and a reference product mAb are challenging for both developers and regulators.
Consequently, in 2014, WHO was requested to update its 2009 SBP Guidelines
to take into account technological advances in the characterization of rDNA-
derived products, and particularly mAbs. In response, WHO organized an
informal consultation in 2015 on the possible amendment of the Guidelines, with
an additional focus placed on SBPs containing mAbs. All participants, including
national regulatory authorities (NRAs) and industry, recognized and agreed
that the evaluation principles described in the WHO Guidelines were still valid,
valuable and applicable in facilitating the harmonization of SBP requirements
globally. It was therefore concluded that there was no need to revise the main
body of the existing WHO Guidelines on SBPs. However, it was also agreed that,
rather than an amendment, there was a need for additional guidance on the
evaluation of biosimilar mAbs.
3. Terminology
The definitions given below apply to the terms as used in these WHO Guidelines.
These terms may have different meanings in other contexts.
American College of Rheumatology 20% improvement criteria
(ACR20): a combined index that measures disease activity in patients with
rheumatoid arthritis, and which corresponds to at least a 20% improvement
in both the tender joint count and the swollen joint count, and at least a 20%
improvement in 3 of 5 other score-set measures.
Antibody-dependent cellular cytotoxicity (ADCC): an immune
mechanism through which Fc receptor-bearing effector cells can recognize
and kill antibody-coated target cells expressing tumour- or pathogen-derived
antigens on their surface.
Antibody-dependent cellular phagocytosis (ADCP): an immune
mechanism which relies on Fc receptors, especially FcγRIIa, on macrophages
or other phagocytic cells which bind to antibodies that are attached to target
cells, followed by the phagocytosis and destruction of target cells, including
tumour cells.
Anti-drug antibodies (ADAs): host antibodies that are capable of
binding to a therapeutic antigen (recombinant protein or mAb). This may or may
not inactivate the therapeutic effects of the treatment and, in rare cases, induce
serious adverse effects.
Area under the curve (AUC): the area under the curve in a plot of
concentration of drug in serum or plasma against time.
AUC t : the area under the concentration-time curve of drug in serum
or plasma from zero up to a definite time t.
AUC tau : the area under the concentration-time curve of drug in serum
or plasma during a dosage interval.
Biological activity: the specific ability or capacity of a product to achieve
a defined biological effect.
Biosimilar mAb: a mAb product that is similar in terms of quality, safety
and efficacy to an already licensed reference product.
C max : the maximum (peak) serum or plasma concentration observed
that a drug achieves in a tested area after the drug has been administered and
prior to the administration of a second dose.
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of cells.
Non-inferiority trial: a trial with the primary objective of showing that
the response to the investigational product is not clinically inferior to that of a
comparative agent.
Overall response rate (ORR): the overall percentage of patients whose
cancer shrinks or disappears after treatment; this includes the rate of complete
response (CR) and partial response (PR).
Potency: the quantitative measure of biological activity based on the
attribute of the product which is linked to the relevant biological properties and
is expressed in units.
Similarity: absence of a relevant difference in the parameter of interest.
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other modifications, could have an impact on the extent of studies needed for
demonstration of biosimilarity. Therefore, the selection of an expression system
for a biosimilar mAb requires careful consideration, with various potential
issues needing to be thoroughly assessed to ensure that an expression system
difference does not result in changes to critical quality attributes.
Since in vitro assays may often be more specific and sensitive for detecting
differences between the SBP and the reference product than studies in animals,
these assays can be considered as paramount for the nonclinical biosimilar
comparability exercise.
■■ Binding studies:
(a) binding to soluble and/or membrane-bound target antigen(s);
and
(b) binding to representative isoforms of the relevant Fc receptors
(that is, for immunoglobulin G (IgG)-based mAbs to FcγRI,
FcγRII and FcγRIII), FcRn and complement (C1q).
These assays are often technically demanding and the models chosen
should be appropriately justified by the applicant (see section 4.1 above). Together,
these assays should broadly cover the functional aspects of the mAb even though
some may not be considered essential for the therapeutic MOA. However, an
evaluation of ADCC, ADCP and CDC may be waived for mAbs directed against
non-membrane-bound targets if appropriately justified.
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5.2.2 General aspects to be considered for all SBPs, including biosimilar mAbs
■■ If there is a need for additional in vivo information, the availability
of a relevant animal species or other relevant models (for example,
transgenic animals or transplant models) should be considered.
■■ If a relevant in vivo animal model is not available the manufacturer
may choose to proceed to human studies, taking into account the
principles for mitigating any potential risk.
■■ When the need for additional in vivo nonclinical studies is evaluated,
the factors to be considered include but are not restricted to:
(a) (the presence of potentially relevant quality attributes that have
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For repeated dose-toxicity studies, where only one dose is evaluated and
the focus of the study is an evaluation of potential qualitative differences in the
toxicity profile between RBP and SBP, the dose would usually be selected at
the high end of the known dosing range of the RBP. Where the focus of the study
is an evaluation of potential quantitative differences with regard to the known
toxicity profile of the RBP, the dose level most likely to reveal differences between
the RBP and SBP should be chosen as justified on the basis of the known toxicity
and/or pharmacodynamic response of the RBP.
The conduct of toxicity studies in non-relevant species (that is, to assess
nonspecific toxicity only, based on impurities) is not recommended. Because
of the different production processes used by the SBP and reference product
manufacturers, qualitative differences in process-related impurities will occur
(for example, host cell proteins). Such impurities should be kept to a minimum
in order to minimize any associated risk.
for which there is little or no experience with the intended clinical route, local
tolerance may need to be evaluated. If other in vivo studies are performed the
evaluation of local tolerance may be part of the design of those studies to avoid
the need for separate local tolerance studies.
6.1.3 Regimen
MAbs are often indicated both for monotherapy and as a part of combination
regimens that incorporate immunosuppressants or chemotherapeutics. It may
be sensible to study the comparative PK in the monotherapy setting in order to
minimize sources of variability. When concomitant therapy alters PK, it may
be appropriate to study comparative PK both in the monotherapy setting and in
combination, particularly where differences cannot be excluded with regard to
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quality attributes that might specifically have an impact on how the drug was
cleared when used in combination.
6.1.5 Doses
A dose should be selected that will enable detection of potential PK differences
between the biosimilar mAb and the reference mAb. MAbs generally possess a
high degree of target selectivity, with many exhibiting nonlinear distribution and
elimination, influenced by binding to their target. In general, it is recommended
that the PK profiles should be compared using the lowest recommended
therapeutic dose. A higher (or the highest) therapeutic dose may be required
where the nonspecific clearance mechanism dominates. For mAbs that are
eliminated by TMD, a low dose (that is, one at which TMD is not saturated) may
be particularly useful for detecting differences in PK (7).
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AUC 0–t , AUC tau , C max and C trough , clearance and half-life. For mAbs that are
administered only intravenously, the aforementioned parameters should be
compared, as should parameters that reflect the clearance of the product.
The clinical data obtained in a sensitive model can also be used to support
extrapolation to other indications of the RBP for which the proposed biosimilar
mAb has not been tested.
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sensitive to detect potential differences between the biosimilar mAb and the
RBP. Hence, historical scientific evidence should be provided which shows that
appropriately designed and conducted trials with the RBP against placebo for
the approved indication have reliably demonstrated the superiority of the RBP
over placebo.
Study population or study end-points may deviate from those which led
to approval of the RBP for the specific indication as long as the primary end-
points are sensitive to the detection of clinically meaningful differences between
the biosimilar mAb and the RBP. Whatever approach is taken, applicants should
always justify their selection of end-points, time points for analysis and the
predefined margin, irrespective of whether this follows the RBP approach or not.
If in doubt, applicants may wish to consult relevant regulatory authorities during
the planning and design stage of the trial.
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The efficacy of the RBP compared to placebo will have been demonstrated
previously. Therefore, it is considered clinically important to ensure that the
biosimilar mAb retains a substantial fraction of the effect of the RBP. As a
consequence, an equivalence margin that preserves a fraction of the smallest
effect size that the RBP can be expected to have relative to a placebo control
is the most suitable. The fraction of the effect size of the RBP that should be
retained by the biosimilar mAb should be clearly justified in each case, and
should take into account the smallest clinically important difference in a given
setting. Once the margin has been selected, determination of the required
sample size should be based on methods specifically designed for equivalence
and non-inferiority trials.
Statistical analysis of data from equivalence trials is typically based on
the indirect confidence interval comparison which requires specification of the
equivalence limits (13). Equivalence is demonstrated when the confidence
interval for the selected metric of the treatment effect falls entirely within
the lower and upper equivalence limits. If a P-value approach is used then the
P-values should be computed on the basis of the two one-sided test (TOST)
procedure, testing simultaneously the null hypotheses of inferiority and
superiority. When using the TOST procedure, equivalence is demonstrated when
the P-values obtained are less than the significance level used.
safety and immunogenicity, and not re-establishing the clinical benefit already
demonstrated by the originator. A surrogate end-point can be used as the primary
end-point when surrogacy to the clinical outcome is well established or generally
accepted, as is the case, for example, with pathological complete response (pCR)
in neoadjuvant treatment of breast cancer. The choice of study end-point should
always be scientifically justified. More sensitive clinical end-points could be used
as secondary end-points for the innovator product, primary or secondary end-
points for the innovator products at different time points of analysis, and/or new
surrogates. For example, overall response rate (ORR) or complete response (CR)
rate can be considered as end-points for clinical efficacy studies of biosimilar
mAbs in oncology trials because these end-points may be more sensitive and
are not time related. However, if progression-free survival (which is one of the
end-points frequently used for clinical efficacy testing for innovator products)
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is considered more sensitive than ORR, then this may be the preferred option.
Likewise, both continuous outcomes (for example, changes in DAS28 from
baseline) and dichotomous outcomes (for example, ACR20) are considered in
rheumatoid arthritis trials for determining clinical comparability (14).
When the primary efficacy end-points that were used for the RBP cannot
be used for the SBP it is advisable to include some common end-points as
secondary end-points to facilitate comparisons between the SBP and the RBP.
The role of these secondary end-points in the overall interpretation of the study
results should be clearly defined, particularly in terms of whether the secondary
end-points are used to support or to confirm equivalency or similarity.
NRAs may not always agree on the choice of study end-points. For an SBP
manufacturer with a global development programme that is guided or required
by various NRAs to fulfil local regulatory or clinical practice requirements it
may be possible to pre-specify different primary study end-points with the
statistical power in the same trial to comply with various regulatory requirements.
6.3.4 Safety
6.3.4.1 General considerations
Comparative safety data should normally be collected pre-authorization. The
extent of data collection depends on the type and severity of known safety
issues for the reference product. The SBP study population should be followed
to provide information on safety events of interest according to experiences
with the reference mAb. Care should be taken to compare the nature, severity
and frequency of adverse events between the biosimilar mAb and the reference
product in clinical trials that enrolled a sufficient number of patients treated
for an acceptable period. Clinical safety issues should be captured throughout
clinical development during initial PK and/or PD evaluations and also in the
primary clinical study establishing comparability.
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■■ the clinically relevant MOA and/or involved receptor(s) are the same;
■■ safety and immunogenicity of the SBP have been sufficiently
characterized and there are no unique or additional safety issues
expected for the extrapolated indication(s).
MAbs have both Fab and Fc-effector functions and may exert their
clinical effect through a variety of mechanisms – for example, ligand blockade,
receptor blockade, receptor down-regulation, cell depletion (via ADCC, CDC
or apoptosis) and signalling induction. A particular mAb may act through one
or a combination of these or other mechanisms. Where a therapeutic mAb is
indicated for a variety of diseases, various MOAs may be important, depending
on the indication in question. In order to support extrapolation, the mechanisms
that contribute to the efficacy of the mAb in each indication should ideally be well
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understood and clearly defined. In practice, this is often not the case. Therefore,
extrapolation may pose additional challenges when a mAb is indicated for a
variety of diseases in which the MOAs are not the same or are not well understood
for each indication. In this situation, it is important to explore the comparability
of in vitro functions of the mAb. In cases where significant functional differences
exist, further nonclinical or clinical data are needed to support extrapolation.
Therefore it is essential that the basic functions of the antibody are considered
when relevant. The tests should be selected according to their relevance for a
particular product and therapeutic indication and, if possible, tailored accordingly
(for example, ADCC assays under different conditions). If minor quality
differences are found, and the affected mechanism is not considered active in the
studied indication, additional steps may be necessary to reach a conclusion on
biosimilarity. Additional data, with appropriate supporting scientific rationale,
could include quality, preclinical and/or PK/PD data and might impact on the
selection of the final clinical, safety and efficacy study. Special post-marketing
measures may be used to monitor aspects of safety and/or immunogenicity in the
extrapolated therapeutic indications.
References
1. Guidelines on evaluation of similar biotherapeutic products (SBPs). In: WHO Expert Committee
on Biological Standardization: sixtieth report. Geneva: World Health Organization; 2013:
Annex 2 (WHO Technical Report Series, No. 977; http://who.int/biologicals/publications/trs/areas/
biological_therapeutics/TRS_977_Annex_2.pdf, accessed 8 December 2016).
2. Guidelines on the quality, safety and efficacy of biotherapeutic protein products prepared by
recombinant DNA technology. In: WHO Expert Committee on Biological Standardization: sixty-
fourth report. Geneva: World Health Organization; 2014: Annex 4 (WHO Technical Report Series,
No. 987; http://www.who.int/biologicals/biotherapeutics/TRS_987_Annex4.pdf?ua=1, accessed
8 December 2016).
3. Thorpe R, Wadhwa M. Intended use of reference products & WHO international standards/
reference reagents in the development of similar biological products (biosimilars). Biologicals.
2011;39(5):262–5 (http://www.sciencedirect.com/science/article/pii/S1045105611000704, accessed
8 December 2016).
4. Preclinical safety evaluation of biotechnology-derived pharmaceuticals. ICH Guideline S6(R1).
Geneva: International Conference on Harmonisation of Technical Requirements for Registration
of Pharmaceuticals for Human Use; 2011 (http://www.ich.org/fileadmin/Public_Web_Site/ICH_
Products/Guidelines/Safety/S6_R1/Step4/S6_R1_Guideline.pdf, accessed 8 December 2016).
5. Wang W, Lu P, Fang Y, Hamuro L, Pittman T, Carr B et al. Monoclonal antibodies with identical
Fc sequences can bind to FcRn differentially with pharmacokinetic consequences. Drug
Metab Dispos. 2011;39(9):1469–77 (http://dmd.aspetjournals.org/content/39/9/1469, accessed
8 December 2016).
125
WHO Expert Committee on Biological Standardization Sixty-seventh report
6. Committee for Medicinal Products for Human Use. Guideline on similar biological medicinal
products containing monoclonal antibodies – non-clinical and clinical issues. London: European
Medicines Agency; 2012 (EMA/CHMP/BMWP/403543/2010; http://www.ema.europa.eu/docs/en_
GB/document_library/Scientific_guideline/2012/06/WC500128686.pdf, accessed 8 December
2016).
7. Pippig SD, Brockmeyer C, Zoubek RE. Biosimilar monoclonal antibodies. In: Dübel S, Reichert JM,
editors. Handbook of therapeutic antibodies. Second edition; pages 681–704. Weinheim: Wiley-
VCH Verlag; 2014; doi: 10.1002/9783527682423.
8. Marini JC, Anderson M, Cai X-Y, Chappell J, Coffey T, Gouty D et al. Systemic verification of
bioanalytical similarity between a biosimilar and a reference biotherapeutic: committee
recommendations for the development and validation of a single ligand-binding assay to
support pharmacokinetic assessments. AAPS J. 2014;16(6):1149–58 (https://www.ncbi.nlm.nih.
gov/pmc/articles/PMC4389757/, accessed 8 December 2016).
9. Guidance for sponsors: information and submission requirements for subsequent entry biologics
(SEBs) [website]. Ottawa: Health Canada; 2010 (http://www.hc-sc.gc.ca/dhp-mps/brgtherap/
applic-demande/guides/seb-pbu/seb-pbu_2010-eng.php, accessed 8 December 2016).
10. Scientific considerations in demonstrating biosimilarity to a reference product. Guidance
for industry. Silver Spring: Food and Drug Administration; 2015 (http://www.fda.gov/
downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM291128.pdf,
accessed 8 December 2016).
11. Committee for Medicinal Products for Human Use. Guideline on similar biological medicinal
products containing biotechnology-derived proteins as active substance: non-clinical and
clinical issues. London: European Medicines Agency; 2015 (EMEA/CHMP/BMWP/42832/2005
Rev1; http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2015/01/
WC500180219.pdf, accessed 9 December 2016).
12. Choice of control group and related issues in clinical trials. E10 (Current Step 4 version). Geneva:
ICH Harmonised Tripartite Guideline. Geneva: International Conference on Harmonisation of
Technical Requirements for Registration of Pharmaceuticals for Human Use; 2000 (http://www.
ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E10/Step4/E10_Guideline.
pdf, accessed 9 December 2016).
13. Chow S-C, Liu J-P. Design and analysis of clinical trials: concepts and methodologies. Second
edition. Hoboken: John Wiley & Sons, Inc.; 2004.
WHO Technical Report Series, No. 1004, 2017
14. Kudrin A, Knezevic I, Joung J, Kang H-N. Case studies on clinical evaluation of biosimilar
monoclonal antibody: scientific considerations for regulatory approval. Biologicals. 2015;43(1):
1–10 (abstract: http://www.sciencedirect.com/science/article/pii/S1045105614001158, accessed
9 December 2016).
15. Wadhwa M, Knezevic I, Kang H-N, Thorpe R. Immunogenicity assessment of biotherapeutic
products: an overview of assays and their utility. Biologicals. 2015;43(5):298–306 (http://www.
sciencedirect.com/science/article/pii/S1045105615000627, accessed 9 December 2016).
16. Knezevic I, Kang H-N, Thorpe R. Immunogenicity assessment of monoclonal antibody products:
a simulated case study correlating antibody induction with clinical outcomes. Biologicals.
2015;43(5):307–17 (http://www.sciencedirect.com/science/article/pii/S1045105615000780,
accessed 9 December 2016).
17. Gupta S, Devanarayan V, Finco D, Gunn GR, Kirshner S, Richards S et al. Recommendations for the
validation of cell-based assays used for the detection of neutralizing antibody immune responses
elicited against biological therapeutics. J Pharm Biomed Anal. 2011;55(5):878–88 (abstract: http://
www.sciencedirect.com/science/article/pii/S0731708511001907, accessed 9 December 2016).
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Guidelines on management of blood and blood
components as essential medicines
1. Introduction 132
2. Purpose and scope 134
3. Blood and blood components as biological therapeutic products 135
3.1 Historical background of blood transfusion 135
3.2 Indications for essential blood and blood component therapy 136
3.3 Risks of blood and blood components 138
4. Preparation of blood and blood components 139
4.1 Ethical aspects of blood donation 139
4.2 Description of key product preparation steps 141
4.3 Associated substances and equipment 145
5. Comparison of blood components with PDMPs 146
5.1 General 146
5.2 Product safety and quality 147
5.3 Product efficacy 148
6. The blood regulatory system 149
6.1 Guiding principles 149
6.2 Regulatory framework 149
6.3 The regulatory authority 153
7. The blood supply system 154
7.1 Organization of the blood supply system 154
7.2 Functions of blood establishments/banks 154
8 The blood transfusion system 154
9. Stepwise implementation of a nationally regulated blood system 155
Authors and acknowledgements 157
References 158
Appendix Examples of existing legislation, regulations and guidance 160
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Abbreviations
EM essential medicine
GMP good manufacturing practice(s)
GPP good preparation practice(s)
GvHD graft versus host disease(s)
HBV hepatitis B virus
HCV hepatitis C virus
HIV human immunodeficiency virus
HLA human leukocyte antigen
NRA national regulatory authority
PDMP plasma-derived medicinal product
PRP platelet-rich plasma
RBC red blood cell
RTTI relevant transfusion-transmitted infection(s)
SOP standard operating procedure
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1. Introduction
Essential medicines (EMs) are defined by WHO as those medicinal products
that satisfy the health-care needs of the majority of the population. They should
therefore be available at all times, in adequate amounts and in appropriate
dosage forms, with assured quality and affordability. The WHO Model List of
Essential Medicines 1 was first generated in 1977 and has been updated every
2 years since then.
This list of EMs includes a core list of minimum medicine needs for a
basic health-care system (that is, the most efficacious, safe and cost-effective
medicines for priority conditions that are selected based on current and estimated
future public health relevance), as well as a complementary list of medicines
for priority diseases for which specialized diagnostic or monitoring facilities,
specialist medical care and/or specialist training are needed. A number of human
plasma-derived medicinal products (PDMPs) – namely, factor VIII concentrate
and factor IX complex concentrate (coagulation factors II, VII, IX and X) – were
added to the 2nd edition of the complementary list of EMs in 1979, followed
by the addition of human normal immunoglobulin to the 15th edition in 2007.
In the 18th edition of the complementary list published in 2013, factor VIII
concentrate and factor IX complex concentrate were replaced with coagulation
factor VIII and coagulation factor IX respectively. Furthermore, anti-D, anti-
rabies and anti-tetanus immunoglobulins were added to the 19th edition of the
core list of EMs in 2015.
In the 2010 World Health Assembly resolution WHA63.12 concern
was expressed about the unequal levels of access globally to blood products,2
particularly PDMPs (also called plasma derivatives). Such inequality of access
left many patients without needed treatment, and many of those with severe
congenital and acquired disorders without adequate plasma-derivative
treatments. In this resolution, the World Health Assembly urged WHO Member
WHO Technical Report Series, No. 1004, 2017
States:
...to take all the necessary steps to update their national regulations on
donor assessment and deferral, the collection, testing, processing, storage,
transportation and use of blood products, and operation of regulatory
authorities in order to ensure that regulatory control in the area of quality
and safety of blood products across the entire transfusion chain meets
internationally recognized standards.
See: http://www.who.int/medicines/publications/essentialmedicines/en/
1
The term “blood products” used in resolution WHA63.12 means blood, blood components and plasma
2
derivatives/PDMPs.
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is also used as a supportive therapy for surgery, chemotherapy, and stem cell
and organ transplantation, as well as the treatment of serious acute and chronic
diseases caused by deficiencies or defects in plasma proteins or cellular blood
components, in order to avoid complications such as life-threatening haemorrhage
or to improve quality of life by reducing anaemia-related symptoms. As blood
systems developed, transfusion evolved from whole blood transfusion to targeted
therapy with specific blood components. This is because several of these diseases
are due to deficiencies or defects in a single blood component or plasma protein
(for example, abnormal or low RBC counts for anaemia (including abnormal
haemoglobin for thalassaemia); low platelet counts for thrombocytopenia; and
clotting factor deficiency for haemophilia). Plasma derived from whole blood or
apheresis can also serve as the starting material for the manufacturing of PDMPs.
In this regard, the transfusion of cellular blood components instead of whole
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Table A3.1
Examples of indications for use of essential blood and blood components
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contains pathogens.
■■ As the collection of blood requires a venepuncture to be performed,
pathogenic bacteria could be transferred into the donation from a
contaminated skin area and subsequently proliferate (particularly in
platelets) to clinically significant numbers capable of causing an RTTI.
This risk must be minimized by the use of standardized and validated
techniques and disinfectants for aseptic venepuncture. Moreover,
thorough adherence to aseptic technique with closed systems and
appropriate microbiological sterility testing should be implemented.
■■ In the case of several pathogens causing severe disease (including
HIV, HBV and HCV) an exposed donor harbouring an RTTI may
feel well and wish to donate despite being at risk of transmitting
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4.2.4 Labelling
The “labelling” of blood and blood components refers to both information
appearing on the direct product label and/or contained in accompanying
documentation. More specifically, the product label should include the product
type, blood groups, a unique donation code that is traceable to the donor, the
site of product preparation, the list of pathogens for which discretionary testing
is performed (for example, cytomegalovirus or hepatitis E virus), the storage
conditions and expiry date (and time of day if applicable). Standardized labels
that can be universally read (such as those printed using machine-readable ISBT
128 standard terminology) should be used. The list of pathogens for which testing
was performed and found to be negative should appear either on the product
label or in accompanying documentation.
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4.2.5 Storage
Blood and blood components should be stored under specified conditions
in order to maintain their safety and quality. Units determined to be safe and
released for transfusion should be segregated from untested units, and access to
storage areas should be restricted to designated personnel. Plasma components
should be frozen within a specified period after collection (preferably within
8 hours for fresh frozen plasma, or within 24 hours). Whole blood and RBCs
should be refrigerated (at 1–6 °C) and platelets should be stored at 20–24 °C
under agitation.
4.2.7 Haemovigilance
4.2.7.1 Documentation
There should be a documentation system that assures bidirectional traceability
of blood components between donors and patients as a foundation of
haemovigilance.
(b) determine whether any donor who contributed to the transfusion is infected
with (or positive for serological markers of) the implicated infectious agent;
(c) trigger a recall of in-date blood or blood components contributed by that
donor; and (d) notify consignees and recipients of components collected from
that donor. The NRA should also be notified as required.
These similarities lead to the underlying concept that blood and blood
components should be prepared within a quality management system based
on the principles of GMP (adapted to blood and blood components) when
relevant and appropriate – and which includes elements such as the testing of
starting materials, in-process quality testing and controls (for example, bacterial
detection and other quality control tests), labelling that reflects product
identity and assures traceability, and adverse event reporting (see section
4.2.8 above). Consequently, the regulation of blood and blood components as
biological therapeutic products would ensure the consistent implementation of
appropriate standards for product quality, safety and efficacy. Such regulation
would apply to all blood establishments/banks involved in the preparation of
these products.
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6.2.2 Legislation
The legislation or law serves as the first level of a comprehensive regulatory
framework and provides a legal basis for the establishment of a regulatory system.
A law is needed that governs the preparation of blood and blood components,
as well as the use of associated substances and relevant medical devices. The
law should define the scope of regulations and provide the legal authority for
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their development. The following are examples of the kinds of provisions that
could be included in legislation:
■■ Definition of the therapeutic products and devices to be regulated.
■■ Prohibitions that prevent the preparation or sale of potentially
unsafe products (for example, products that are adulterated or
prepared under unsanitary conditions).
■■ Assignment of an NRA with legal powers to administer, enforce
and verify compliance with the legislation and regulations (for
example, powers for inspection, seizure and forfeiture and for the
establishment of a list that sets out the classes of products to be
regulated).
■■ The offences and punishment of persons who deliberately
contravene the legislation or regulations.
■■ Definition of the areas for which regulations should be developed
and granting of the authority to develop the regulations necessary
for carrying the purposes and provisions of the legislation into effect.
These areas should include those covered below in section 6.2.3.
Detailed guidance regarding provisions that could be included in
national acts or legislation may be found in the list of documents provided in
the Appendix to these Guidelines.
6.2.3 Regulations
Regulations form the second level of the regulatory framework. They are
developed under the authority of the legislation and serve to interpret the
legislation and to provide policies and standards/technical requirements that are
legally binding.
WHO Technical Report Series, No. 1004, 2017
and relevant medical devices (such as in vitro screening and diagnostic test kits,
blood-collection equipment and blood bag systems) that are used during the
preparation of blood and blood components. Systems should be put in place to
ensure compliance with these regulations.
The blood transfusion system consists of care centres (hospitals, surgical centres
and outpatient facilities; and sometimes ambulances) that utilize blood and
blood components for the treatment of patients. Such centres are responsible for
carrying out the following activities:
■■ storing blood and blood components at appropriate temperatures
and conditions;
■■ developing appropriate procedures for further processing of the
blood and blood components prior to transfusion – for example,
pooling, washing and irradiation, where applicable;
■■ appropriate pre-transfusion testing of patients and cross-matching
to ensure compatibility of the blood component to be transfused;
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be involved from the beginning in order to understand and define their
individual responsibilities and expected contributions. Regular interaction
among stakeholders is essential.
The legislative body should define a legal framework (regulation)
applicable to blood and blood components. This would include assigning the
NRA to oversee all institutions and health-care professionals supplying blood
and blood components.
An NRA is an essential element of a regulatory system. The decision to
adopt a particular regulatory model should take into account existing regulatory
structures, capacities and expertise. Establishing the regulation of blood and
blood components under the NRA for medicines may be the most effective
and rapid way to accomplish this in settings where blood regulation is otherwise
lacking. Regulatory frameworks for blood and blood components and for PDMPs
should be complementary.
Blood establishments, other related health institutions, and health-care
professionals supplying blood and blood components for transfusion should
be engaged and their experience used to inform the establishment of standards
and procedures. This may result in improving the existing structure of the blood
system. The initial use of existing standards as a starting point for establishing a
common language between all key players may provide an acceptable approach
for all parties.
Where applicable, representatives of the plasma fractionation industry
should be invited to participate as additional key players to support this process.
This should ensure that appropriate standards are implemented and that the
quality of surplus plasma as a starting material for further manufacturing will
meet the necessary requirements.
The development of national blood standards covering donor-selection
criteria, infectious disease marker testing strategies, quality system requirements
and standards for the final products (specifications and/or monographs) will
WHO Technical Report Series, No. 1004, 2017
be an essential step. Both during and after the legislative process, these initial
standards may continuously be improved and implemented on a national
basis. The establishment of such standards should take into account existing
national and international guidelines such as the WHO guidelines on good
manufacturing practices for blood establishments (2). As soon as possible,
blood establishments should apply these standards in a consistent manner by
implementing appropriate procedures (including SOPs and training) within
their quality system.
A parallel national implementation process and regular interaction
among the key players will be essential in accelerating the implementation
of standards and regulatory functions. Since the process of implementing a
regulatory system and reaching an acceptable compliance status may take
several years, a political decision will be required to define the time frames
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for reaching full compliance with standards and for effective enforcement
of regulations by the NRA. A possible stepwise implementation plan for a
nationally regulated blood system is outlined in Fig. A3.1.
Fig. A3.1
Stepwise implementation plan for a nationally regulated blood system
References
1. Assessment criteria for national blood regulatory systems. Geneva: World Health Organization;
2012 (http://www.who.int/bloodproducts/NationalBloodRegSystems.pdf, accessed 26 December
2016).
2. WHO guidelines on good manufacturing practices for blood establishments. In: WHO Expert
Committee on Specifications for Pharmaceutical Preparations: forty-fifth report. Geneva: World
Health Organization; 2011: Annex 4 (WHO Technical Report Series, No. 961; http://www.who.int/
bloodproducts/publications/GMP_Bloodestablishments.pdf?ua=1, accessed 26 December 2016).
3. Highlights of transfusion medicine history [website]. Bethesda (MD): American Association of
Blood Banks (http://www.aabb.org/tm/Pages/highlights.aspx, accessed 26 December 2016).
4. History of blood transfusion [website]. Washington (DC): American Red Cross (http://www.
redcrossblood.org/learn-about-blood/blood-transfusions/history-blood-transfusions, accessed
26 December 2016).
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5. Blood donor selection: guidelines on assessing donor suitability for blood donation. Geneva:
World Health Organization; 2012; chapter 7 (http://www.who.int/bloodsafety/publications/
guide_selection_assessing_suitability.pdf, accessed 26 December 2016).
6. WHO guidelines on drawing blood: best practices in phlebotomy. Geneva: World Health
Organization; 2010 (http://www.who.int/injection_safety/phleb_final_screen_ready.pdf, accessed
26 December 2016).
7. ‘Sterility testing of blood components and advanced therapy medicinal products’ (Munich,
April 29, 2010) organized by the DGTI section ‘Safety in hemotherapy’ – Abstracts. Transfus
Med Hemother. 2011;38(5):337–40 (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3364038,
accessed 26 December 2016).
8. Bacterial risk control strategies for blood collection establishments and transfusion services to
enhance the safety and availability of platelets for transfusion. Silver Spring (MD): US Food and
Drug Administration; 2016 (http://www.fda.gov/downloads/Guidances/Blood/UCM425952.pdf,
accessed 26 December 2016).
9. Blood basics [website]: Washington (DC): American Society of Hematology (http://www.
hematology.org/Patients/Basics/, accessed 26 December 2016).
10. What does blood do? [website]. Bethesda (MD): PubMed Health (http://www.ncbi.nlm.nih.gov/
pubmedhealth/PMH0072576/, accessed 26 December 2016).
11. Circular of Information for the use of human blood and blood components. Revised November
2013. Bethesda (MD): American Association of Blood Banks (https://www.aabb.org/tm/coi/
Documents/coi1113.pdf, accessed 26 December 2016).
12. Circular of Information for the use of human blood components. Ottawa: Canadian Blood
Services (https://www.blood.ca/en/hospitals/circular-information, accessed 26 December 2016).
13. Folléa G. Donor compensation and remuneration – is there really a difference? ISBT Science
Series. 2016;11(S1):3–9 (http://onlinelibrary.wiley.com/doi/10.1111/voxs.12188/full, accessed
26 December 2016).
14. Eligibility for blood donation: recommendations for education and selection of prospective
blood donors. Washington (DC): Pan American Health Organization; 2009 (http://www1.paho.
org/hq/dmdocuments/2009/EligiBlood09EN.pdf?ua=1, accessed 26 December 2016).
15. Guidelines on estimation of residual risk of HIV, HBV or HCV infections via cellular blood
components and plasma. In: WHO Expert Committee on Biological Standardization: sixty-seventh
report. Geneva: World Health Organization; 2017: Annex 4 (WHO Technical Report Series, 1004).
159
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Appendix
Examples of existing legislation, regulations and guidance
The documents listed here are provided as examples of existing legislation,
regulations and guidance that may be helpful in establishing a national
regulatory framework.
http://laws-lois.justice.gc.ca/eng/regulations/C.R.C.,_c._870/
index.html (accessed 26 December 2016).
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Table continued
Country Relevant legislation, regulations and guidance
or region
Europe 1. Directive 2002/98/EC of the European Parliament and of the
Council of 27 January 2003 – setting standards of quality
and safety for the collection, testing, processing, storage and
distribution of human blood and blood components and
amending Directive 2001/83/EC: https://ec.europa.eu/health/sites/
health/files/files/eudralex/vol-1/dir_2002_98/dir_2002_98_en.pdf
(accessed 26 December 2016).
2. Commission Directive 2004/33/EC of 22 March 2004 –
implementing Directive 2002/98/EC of the European Parliament
and of the Council as regards certain technical requirements
for blood and blood components: http://www.ema.europa.eu/
docs/en_GB/document_library/Regulatory_and_procedural_
guideline/2009/10/WC500004484.pdf (accessed 26 December
2016).
3. Commission Directive 2005/62/EC of 30 September 2005 –
implementing Directive 2002/98/EC of the European Parliament
and of the Council as regards Community standards and
specifications relating to a quality system for blood establishments:
http://www.ema.europa.eu/docs/en_GB/document_library/
Regulatory_and_procedural_guideline/2009/10/WC500004486.pdf
(accessed 26 December 2016).
4. Commission Directive 2005/61/EC of 30 September 2005 –
implementing Directive 2002/98/EC of the European Parliament
and of the Council as regards traceability requirements and
notification of serious adverse reactions and events: http://www.
ema.europa.eu/docs/en_GB/document_library/Regulatory_and_
procedural_guideline/2009/10/WC500004485.pdf (accessed
26 December 2016).
5. Council of Europe (EDQM) Recommendations & Resolutions:
https://www.edqm.eu/en/blood-transfusion-recommendations-
resolutions-71.html (accessed 26 December 2016).
The USA 1. United States Food and Drug Administration; subchapter F –
Biologics: http://www.ecfr.gov/cgi-bin/text-idx?SID=7f01bf
1d25c364e2d287f227cd6833c8&mc=true&tpl=/ecfrbrowse/
Title21/21CIsubchapF.tpl (accessed 26 December 2016).
2. United States Food and Drug Administration; Blood
Guidances: http://www.fda.gov/BiologicsBloodVaccines/
GuidanceComplianceRegulatoryInformation/Guidances/Blood/
default.htm (accessed 26 December 2016).
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Guidelines on estimation of residual risk of HIV, HBV or
HCV infections via cellular blood components and plasma
1. Introduction 166
2. Purpose and scope 166
3. Terminology 167
4. Course of HIV, HBV and HCV infections 170
4.1 Acute infection 170
4.2 Chronic persistent infection 170
5. Residual risk origins 171
5.1 Assay failures 171
5.2 Diagnostic window periods 172
6. Screening assay categories and diagnostic window periods 173
6.1 Screening assay categories 173
6.2 Diagnostic window periods 175
7. Virus concentrations during diagnostic window period 178
8. Confirmation of reactive screening results 178
9. Virus epidemiology of donor populations 179
9.1 First-time donors 179
9.2 Repeat donors 180
10. Estimation of incidence and window period modelling of risks 180
10.1 Incidence 180
10.2 Residual risk per blood donation in repeat donors 181
11. Residual risks 183
11.1 Infection of recipients of non-pathogen-inactivated blood components 184
11.2 Contamination of plasma pools 184
Authors and acknowledgements 185
References 186
Appendix 1 Evaluation of new blood-screening assays 190
Appendix 2 Examples for estimation of residual risks 194
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Abbreviations
anti-HBc antibodies to hepatitis B core protein
anti-HBs antibodies to hepatitis B surface antigen
CE Conformité Européenne (conforms to European requirements)
CLIA chemiluminescence immunoassay
EIA enzyme immunoassay
FDA Food and Drug Administration
HBsAg hepatitis B surface antigen
HBV hepatitis B virus
HCV hepatitis C virus
HIV human immunodeficiency virus
ID-NAT individual donation nucleic acid amplification technique
IDI interdonation interval
IU International Unit(s)
IVD in vitro diagnostic
MP-NAT minipool nucleic acid amplification technique
NAT nucleic acid amplification technique
OBI occult hepatitis B infection
P probability
PCR polymerase chain reaction
PDMP plasma-derived medicinal product
RDT rapid diagnostic test
RR residual risk (used in mathematical formulae)
vDWP viraemic phase of the diagnostic window period
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1. Introduction
The course that a viral infection may take in an individual and the different
phases of viral infections are described in the following sections – together with
the advantages and limitations of using different blood-screening assays for the
different infection phases. Blood-screening assays are differentiated by distinct
categories. The residual risk of missing viral infections using any screening
assay is mainly due to the viraemic phase of the diagnostic window period
(vDWP) for each assay – the mean size of which varies between different assay
categories. Another component of the residual risk is the virus epidemiology
of the donor population (consisting of repeat and first-time donors) with the
rate of new infections (incidence) in donors determining the probability of
window-period donations. The residual risk per donation from the repeat-
donor subpopulation may be used to extrapolate the respective risk for the first-
time donor subpopulation, for which incidence data are often unavailable. The
residual risk affects recipients of non-pathogen-inactivated blood components to
whom viruses may be transmitted. It also determines the potential viral load of
plasma pools used for the manufacturing of plasma-derived medicinal products
(PDMPs); this potential contamination level needs to be assessed against the
viral inactivation or reduction strategies in the manufacturing process.
in the prevalence and incidence of viral infections in blood donors around the
world. The impact of such epidemiological differences on blood safety needs
to be assessed together with the sensitivity of the testing strategy applied. Such
assessments may be used to guide strategic decisions on the choice of assays to
detect virus-positive blood donations and as a basis for cost–benefit analysis of
the different testing scenarios most suitable in the region. The factors influencing
the risk of virus transmission by blood components are described, as well as
simple mathematical formulae to calculate its probability. These estimates may
also be used to counsel recipients on the risks of transfusion. Similarly, the
probability and potential level of viral contamination of plasma pools used
for the manufacture of PDMPs can be calculated. The infectivity risk of plasma
products can then be estimated in relation to the inactivation and reduction
capacity of the manufacturing process.
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3. Terminology
The definitions given below apply to the terms as used in these WHO Guidelines.
These terms may have different meanings in other contexts.
Analytical sensitivity: the smallest amount of the target marker that
can be precisely detected by an IVD assay; it may be expressed as the limit of
detection and is often determined by testing limiting dilutions of a biological
reference preparation.
Apheresis: the process by which one or more blood components are
selectively obtained from a donor by withdrawing whole blood, separating it
by centrifugation and/or filtration into its components, and returning those
not required to the donor. The term “plasmapheresis” is used for a procedure
dedicated specifically to the collection of plasma.
Blood collection: a procedure whereby a single donation of blood is
collected in a sterile receptacle containing anticoagulant and/or stabilizing
solution, under conditions designed to minimize microbiological contamination,
cellular damage and/or coagulation activation.
Blood component: a constituent of blood that can be used directly or
after further processing for therapeutic applications. The main therapeutic blood
components are red blood cell concentrates, platelet concentrates, plasma for
transfusion and cryoprecipitate.
Blood establishment: any structure, facility or body that is responsible
for any aspect of the collection, testing, processing, storage, release and/or
distribution of human blood or blood components when intended for transfusion
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NAT conversion: the time period during which specific nucleic acids
(for example, viral nucleic acids after a recent virus infection) become detectable
by a nucleic acid amplification technique.
Nucleic acid amplification technique (NAT): a testing method to detect
the presence of a targeted area of a defined nucleic acid sequence (for example,
viral genome) using amplification techniques such as polymerase chain reaction
(PCR) or transcription mediated amplification (TMA).
Plasma: the liquid portion remaining after separation of the cellular
elements from blood – collected in a receptacle containing an anticoagulant, or
separated by the continuous filtration or centrifugation of anticoagulated blood.
Plasma for fractionation: plasma (from whole blood or apheresis) used
for the production of PDMPs.
Plasma for transfusion: plasma (from whole blood or apheresis) used
for direct infusion into patients without a prior fractionation step. It can be
subjected to treatment for inactivating a broad range of pathogens.
Plasma-derived medicinal products (PDMPs): a range of medicinal
products obtained by the fractionation of human plasma. Also called plasma
derivatives, plasma products or fractionated plasma products.
Plasmapheresis: see “Apheresis” above.
Prevalence: the proportion of past infections identified over a specified
period in a defined population.
Recovered plasma: plasma recovered from a whole blood donation and
used for transfusion or for fractionation into PDMPs.
Repeat donor: a person who has donated blood/plasma previously in the
blood establishment. The definition of this term may differ between legislations.
Sensitivity: see “Analytical sensitivity” and “Diagnostic sensitivity” above.
Seroconversion: the time period during which specific antibodies
develop (for example, after a recent virus infection) and become detectable in
the blood; this term is sometimes also used for the time period during which
viral antigens, such as hepatitis B surface antigen (HBsAg), or viral nucleic
acids become detectable in the blood after recent infection. See also “NAT
conversion” above.
Source plasma: plasma obtained by apheresis for further fractionation
into PDMPs.
Viraemic phase of the diagnostic window period (vDWP): the part of
the diagnostic window period during which viruses are present in the blood; the
beginning of the viraemic phase is defined by the putative presence of one virus
particle in a blood component (20 mL plasma for packed red blood cells) and
can be extrapolated using viral replication kinetics (viral doubling time).
Window period: see “Diagnostic window period” above.
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length of viraemia detection is longer. The transient nature of these HBV blood-
screening markers requires the use of an adjustment factor when calculating
rates of new infections (1).
window period is relevant. The start of the potentially infectious window period
during the early ramp-up phase of viraemia can be defined as the point at
which one virus particle is present in a blood component. A generally accepted
worst-case assumption for cellular components is to define the start of the
infectious window period as the point at which the concentration reaches one
virus particle in 20 mL of plasma (the volume co-transfused with a red blood
cell unit suspended in additive solution) (14). Viral replication characteristics
in the early phase of infection are rather consistent among recently infected
individuals. This phenomenon results in characteristic doubling times of plasma
viral concentration for HIV, HBV and HCV. By knowing the viral replication
kinetics of HIV, HBV or HCV in the early infection phase, along with the
diagnostic sensitivity of the screening assay, the length of the viraemic phase can
be extrapolated for each screening assay.
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5.2.1 HIV
HIV replicates with an average doubling time of 20 hours (0.83 days) to
reach a peak level of viraemia of up to 10 7 IU HIV RNA/mL (15). This virus
concentration decreases in parallel with the development of specific antibodies
detectable by anti-HIV assays. The currently most sensitive antigen assays
can detect HIV p24 antigen at a level corresponding to 10 4 IU HIV RNA/mL.
Most HIV antigen‑antibody combination (“combo”) assays are less sensitive
in their detection of p24 antigen when compared to antigen assays – with
the corresponding HIV RNA concentration for detection by state-of-the-art
combo assays being around 10 5 IU/mL (15, 16). Attention should be paid to
donors having taken early antiretroviral treatment or pre-exposure antiretroviral
treatment which could reverse seroconversion and lower viral load (17).
5.2.2 HBV
The replication rate of HBV in the early infection phase as determined by the
increase in viraemia is significantly lower when compared to HIV or HCV,
with an HBV average doubling time of 2.6 days (18, 19). HBV viraemia in the
early infection phase is detected earlier by NAT-based assays than by HBsAg
assays. In the absence of NAT-based assays the use of HBsAg assays with a high
analytical sensitivity is key for the detection of early infection.
5.2.3 HCV
For HCV an average doubling time of 10.8 hours (0.45 days) during the ramp‑up
phase has been determined, followed by an anti-HCV-negative plateau phase
of several weeks characterized by high-level viraemia of up to 10 8 IU HCV
RNA/mL (20, 21). HCV core antigen appears to be detectable by core antigen
assays during the major part of this anti-HCV-negative phase, namely the
entire plateau phase and the last part of the ramp-up phase. Similar to HIV, the
antigen detection efficiency of current HCV combo assays is less than that of
the antigen assays. Combo assays have an overall detection rate of approximately
40% of anti-HCV-negative window period specimens, and preferentially detect
those with virus concentrations above 10 6 IU HCV RNA/mL (22).
■■ antigen
■■ combo
■■ antibody
■■ rapid diagnostic test (RDT).
While antibody assays are designed to detect both recent and chronic
persistent infections, the additional benefit of antigen or viral genome detection
lies mainly in further reducing the diagnostic window period. The length of the
diagnostic window period varies greatly between the different assay categories.
6.1.5 RDTs
RDTs are diagnostic devices of simple design, often based on
immunochromatographic (lateral flow) or immunofiltration (flow-through)
technologies. RDTs do not require complex equipment and provide the test
result within a short time (15–30 minutes). Although often not claimed by the
manufacturer a suitable for use in blood screening, these devices are sometimes
used for blood-safety testing in resource-limited settings or in emergency
situations. RDT technology is associated with a lower sensitivity than that of
more sophisticated immunoassays developed specifically for blood screening
(25, 26).
have differing sensitivities for different viral genotypes and/or for viral subtypes.
The vast majority of commercial seroconversion panels used for diagnostic
sensitivity studies originate from regular plasma donors, and mainly represent
viral genotypes and subtypes prevalent in Europe and the United States (namely
HIV subtype B, HCV genotypes 1–3 and HBV genotype A). However, the
sensitivity of assays observed with these seroconversion panels may not always
be representative for early infection with viral genotypes prevalent elsewhere in
the world (27). Further details on this and other considerations in the evaluation
of new blood-screening assays are provided in Appendix 1.
Mean estimates of the length of the vDWP for so-called state-of-the-
art assays are presented by assay category in Table A4.1. These estimates should
be used for risk calculation unless more detailed information is available on
the sensitivity and corresponding window period of the assay used for blood
screening. Hence, if comparative data obtained with multiple seroconversion
panels indicate that the sensitivity of a specific assay is clearly different from the
mean value shown in Table A4.1, the more accurate data for this assay should be
used for the estimation of residual risk.
WHO Technical Report Series, No. 1004, 2017
176
Table A4.1
Length of the vDWP for different assay categories (days) a
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Table A4.2
Maximal concentration of viral genomes in the vDWP (IU/mL) a
a mean detection period for the transient HBV marker (HBsAg or HBV DNA)
can be factored into the adjustment. Further contributions to the adjustment
factor originate from HBV infections without detectable antigenaemia (assumed
to be 25%; transiently picked up by sensitive HBV NAT-based assays) (1). The
scientific literature provides several different estimates for the length of transient
antigenaemia (1, 19, 41). The differences observed between the underlying
studies may be explained by different infection routes, different inoculum,
different HBV genotypes, and HBsAg or HBV-DNA assays of different sensitivity.
The lengths of the HBV marker detection periods have been estimated
from the available data for the different assay categories and are listed in
Table A4.3.
Table A4.3
HBV DNA and HBsAg detection period (days) for different assay categories
IDI
The HBV incidence adjustment factor is calculated as 100/P. For results where
P ≥ 100% no adjustment is necessary.
To determine the RR per donation for HBV infection, the figure obtained
for HBV using Formula 2 in section 10.2 above is then multiplied by the
adjustment factor for the specific assay category used.
If, however, only a few acute infections are found it is advised to take the average
IDI of all repeat donors.
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References
1. Korelitz JJ, Busch MP, Kleinman SH, Williams AE, Gilcher RO, Ownby HE et al. A method for estimating
hepatitis B virus incidence rates in volunteer blood donors. Transfusion. 1997;37(6):634–40
(https://www.researchgate.net/publication/14027062_A_method_for_estimating_hepatitis_B_
virus_incidence_rates_in_volunteer_blood_donors_National_Heart_Lung_and_Blood_
Institute_Retrovirus_Epidemiology_Donor_Study, accessed 18 December 2016).
2. Guidelines for the screening, care and treatment of persons with chronic hepatitis C infection.
Updated version April 2016. Geneva: World Health Organization; 2016 (http://apps.who.int/iris/
bitstream/10665/205035/1/9789241549615_eng.pdf?ua=1, accessed 18 December 2016).
3. Hollinger FB. Hepatitis B virus infection and transfusion medicine: science and the occult.
Transfusion. 2008;48(5):1001–26.
4. Allain JP. Occult hepatitis B virus infection: implications in transfusion. Vox Sang. 2004;86(2):83–91
(abstract: https://www.ncbi.nlm.nih.gov/pubmed/15023176/, accessed 18 December 2016).
5. Seed CR, Maloney R, Kiely P, Bell B, Keller AJ, Pink J. Infectivity of blood components from
donors with occult hepatitis B infection – results from an Australian lookback programme. Vox
Sang. 2015;108(2):113–22 (abstract: https://www.ncbi.nlm.nih.gov/pubmed/25234417, accessed
18 December 2016).
6. Allain JP, Mihaljevic I, Gonzalez-Fraile MI, Gubbe K, Holm-Harritshøj L, Garcia JM et al. Infectivity of
blood products from donors with occult hepatitis B virus infection. Transfusion. 2013;53(7):1405–
15 (abstract: https://www.ncbi.nlm.nih.gov/pubmed/23362802, accessed 18 December 2016).
7. Satake M, Taira R, Yugi H, Hino S, Kanemitsu K, Ikeda H et al. Infectivity of blood components with
low hepatitis B virus DNA levels identified in a lookback program. Transfusion. 2007;47(7):1197–
205 (abstract: https://www.ncbi.nlm.nih.gov/pubmed/17581154, accessed 18 December 2016).
8. Levicnik-Stezinar S, Rahne-Potokar U, Candotti D, Lelie N, Allain JP. Anti-HBs positive occult
hepatitis B virus carrier blood infectious in two transfusion recipients. J Hepatol. 2008;48(6):1022–5
(https://www.researchgate.net/publication/5417215_Anti-HBs_positive_occult_hepatitis_B_
virus_carrier_blood_infectious_in_two_transfusion_recipients, accessed 18 December 2016).
9. Spreafico M, Berzuini A, Foglieni B, Candotti D, Raffaele L, Guarnori I et al. Poor efficacy of
nucleic acid testing in identifying occult HBV infection and consequences for safety of blood
supply in Italy. J Hepatol. 2015;63(5):1068–76 (abstract: https://www.researchgate.net/
WHO Technical Report Series, No. 1004, 2017
publication/279300519_Poor_efficacy_of_nucleic_acid_testing_in_identifying_occult_HBV_
infection_and_consequences_for_safety_of_blood_supply_in_Italy, accessed 18 December
2016).
10. Humpe A, Legler TJ, Nübling CM, Riggert J, Unger G, Wolf C et al. Hepatitis C virus transmission
through quarantine fresh-frozen plasma. Thromb Haemost. 2000;84(5):784–8 (abstract: https://
www.researchgate.net/publication/12200759_Hepatitis_C_virus_transmission_through_
quarantine_fresh-frozen_plasma, accessed 18 December 2016).
11. Schmidt M, Korn K, Nübling CM, Chudy M, Kress J, Horst HA et al. First transmission of human
immunodeficiency virus Type 1 by a cellular blood product after mandatory nucleic acid
screening in Germany. Transfusion. 2009;49(9):1836–44 (abstract: https://www.researchgate.
net/publication/24438716_First_transmission_of_HIV-1_by_a_cellular_blood_product_after_
mandatory_NAT_blood_screening_in_Germany, accessed 19 December 2016).
12. Chudy M, Weber-Schehl M, Pichl L, Jork C, Kress J, Heiden M et al. Blood screening nucleic
acid amplification tests for human immunodeficiency virus Type 1 may require two different
amplification targets. Transfusion. 2012;52(2):431–9 (abstract: https://www.researchgate.net/
186
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publication/51541171_Blood_screening_nucleic_acid_amplification_tests_for_human_
immunodeficiency_virus_Type_1_may_require_two_different_amplification_targets, accessed
19 December 2016).
13. Post-market surveillance for in vitro diagnostics (IVDs) [website]. Geneva: World Health
Organization (http://www.who.int/diagnostics_laboratory/postmarket/en/, accessed 19 December
2016).
14. Busch MP, Glynn SA, Stramer SL, Strong DM, Caglioti S, Wright DJ et al. A new strategy for estimating
risks of transfusion-transmitted viral infections based on rates of detection of recently infected
donors. Transfusion. 2005;45(2):254–64 (https://www.researchgate.net/publication/8070296_A_
new_strategy_for_estimating_risk_of_transfusion-transmitted_viral_infections_based_on_
rates_of_detection_of_recently_infected_donors, accessed 19 December 2016).
15. Fiebig EW, Wright DJ, Rawal BD, Garrett PE, Schumacher RT, Peddada L et al. Dynamics of
HIV viremia and antibody seroconversion in plasma donors: implications for diagnosis and
staging of primary HIV infection. AIDS. 2003;17(13):1871–9 (https://www.researchgate.net/
publication/271733355_Dynamics_of_HIV_viremia_and_antibody_seroconversion_in_plasma_
donors, accessed 20 December 2016).
16. Consolidated guidelines on HIV testing services. Geneva: World Health Organization; 2015
(http://apps.who.int/iris/bitstream/10665/179870/1/9789241508926_eng.pdf?ua=1&ua=1,
accessed 20 December 2016).
17. de Souza MS, Pinyakorn S, Akapirat S, Pattanachaiwit S, Fletcher JLK, Chomchey N et al.
Initiation of antiretroviral therapy during acute HIV-1 infection leads to a high rate of
nonreactive HIV serology. Clin Infect Dis. 2016;63(4):555–61 (https://www.researchgate.net/
publication/304070497_Initiation_of_Antiretroviral_Therapy_during_Acute_HIV-1_Infection_
leads_to_A_High_Rate_of_Non-Reactive_HIV_Serology, accessed 20 December 2016).
18. Biswas R, Tabor E, Hsia CC, Wright DJ, Laycock ME, Fiebig EW et al. Comparative sensitivity of HBV
NATs and HBsAg assays for detection of acute HBV infection. Transfusion. 2003;43(6):788–98
(https://www.researchgate.net/publication/10750139_Comparative_sensitivity_of_HBVNATs_
and_HBsAg_assays_for_detection_of_acute_HBV_infection, accessed 20 December 2016).
19. Yoshikawa A, Gotanda Y, Itabashi M, Minegishi K, Kanemitsu K, Nishioka K. Hepatitis B NAT virus-
positive blood donors in the early and late stages of HBV infection: analyses of the window
period and kinetics of HBV DNA. Vox Sang. 2005;88(2):77–86. Erratum in Vox Sang. 2005;88(3):223
(https://www.ncbi.nlm.nih.gov/pubmed/15720604/, accessed 20 December 2016).
20. Nübling CM, Unger G, Chudy M, Raia S, Löwer J. Sensitivity of HCV core antigen and HCV RNA
detection in the early infection phase. Transfusion. 2002;42(8):1037–45 (abstract: https://www.
researchgate.net/publication/11076628_Sensitivity_of_HCV_core_antigen_and_HCV_RNA_in_
early_infection_phase, accessed 20 December 2016).
21. Glynn SA, Wright DJ, Kleinman SH, Hirschkorn D, Tu Y, Heldebrant C et al. Dynamics of viremia
in early hepatitis C virus infection. Transfusion. 2005;45(6):994–1002 (https://www.researchgate.
net/publication/7807301_Dynamics_of_viremia_in_early_hepatitis_C_virus_infection, accessed
20 December 2016).
22. Laperche S, Nübling CM, Stramer SL, Brojer E, Grabarczyk P, Yoshizawa H et al. Sensitivity
of hepatitis C virus core antigen and antibody combination assays in a global panel of
window period samples. Transfusion. 2015;55(10):2489–98 (https://www.researchgate.net/
publication/277412688_Sensitivity_of_hepatitis_C_virus_core_antigen_and_antibody_
combination_assays_in_a_global_panel_of_window_period_samples_HCV_Core_Antigen_
Detection_in_Window_Period, accessed 20 December 2016).
187
WHO Expert Committee on Biological Standardization Sixty-seventh report
23. Weusten JJ, van Drimmelen HA, Lelie N. Mathematic modeling of the risk of HBV, HCV, and HIV
transmission by window-phase donations not detected by NAT. Transfusion. 2002;42(5):537–48
(abstract: https://www.researchgate.net/publication/11291469_Mathematic_modeling_of_the_
risk_of_HBV_HCV_and_HIV_transmission_by_window-phase_donations_not_detected_by_
NAT, accessed 20 December 2016).
24. Weusten J, Vermeulen M, van Drimmelen H, Lelie N. Refinement of a viral transmission risk
model for blood donations in seroconversion window phase screened by nucleic acid testing in
different pool sizes and repeat test algorithms. Transfusion. 2011;51(1):203–15 (abstract: https://
www.ncbi.nlm.nih.gov/pubmed/20707858, accessed 20 December 2016).
25. Scheiblauer H, El-Nageh M, Diaz S, Nick S, Zeichhardt H, Grunert H-P et al. Performance
evaluation of 70 hepatitis B virus (HBV) surface antigen (HBsAg) assays from around the world by
a geographically diverse panel with an array of HBV genotypes and HBsAg subtypes. Vox Sang.
2010;98(3p2):403–14 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860763/, accessed 20
December 2016).
26. Scheiblauer H, El-Nageh M, Nick S, Fields H, Prince A, Diaz S. Evaluation of the performance
of 44 assays used in countries with limited resources for the detection of antibodies to
hepatitis C virus. Transfusion. 2006;46(5):708–18 (abstract: https://www.ncbi.nlm.nih.gov/
pubmed/16686838, accessed 20 December 2016).
27. Apetrei C, Loussert-Ajaka I, Descamps D, Damond F, Saragosti S, Brun-Vézinet F et al. Lack of
screening test sensitivity during HIV-1 non-subtype B seroconversions. AIDS. 1996;10(14):F57–60
(abstract: https://www.ncbi.nlm.nih.gov/pubmed/8970678, accessed 20 December 2016).
28. 2009/108/EC: Commission Decision of 3 February 2009 amending Decision 2002/364/EC on
common technical specifications for in vitro-diagnostic medical devices. European Commission.
Official Journal of the European Union. 2009;L39/34–L39/49 (http://eur-lex.europa.eu/legal-
content/EN/TXT/?uri=uriserv:OJ.L_.2009.039.01.0034.01.ENG&toc=OJ:L:2009:039:TOC, accessed
18 December 2016).
29. Assal A, Barlet V, Deschaseaux M, Dupont I, Gallian P, Guitton C et al. Sensitivity of two hepatitis B
virus, hepatitis C virus (HCV), and human immunodeficiency virus (HIV) nucleic acid test systems
relative to hepatitis B surface antigen, anti-HCV, anti-HIV, and p24/anti-HIV combination assays in
seroconversion panels. Transfusion. 2009;49(2):301–10 (abstract: https://www.ncbi.nlm.nih.gov/
pubmed/19389212, accessed 20 December 2016).
30. Kleinman SH, Lelie N, Busch MP. Infectivity of human immunodeficiency virus-1, hepatitis C
WHO Technical Report Series, No. 1004, 2017
virus, and hepatitis B virus and risk of transmission by transfusion. Transfusion. 2009;49(11):
2454–89 (available at: https://www.researchgate.net/publication/26743554_Infectivity_of_
human_immunodeficiency_virus-1_hepatitis_C_virus_and_hepatitis_B_virus_and_risk_of_
transmission_by_transfusion, accessed 20 December 2016).
31. Screening donated blood for transfusion-transmissible infections. Recommendations. Geneva:
World Health Organization; 2010 (http://www.who.int/bloodsafety/ScreeningDonatedBloodfor
Transfusion.pdf, accessed 20 December 2016).
32. Murphy G, Parry JV. Assays for the detection of recent infections with human immunodeficiency
virus type 1. Euro Surveill. 2008;13(36):4–10 (http://www.eurosurveillance.org/ViewArticle.
aspx?ArticleId=18966, accessed 20 December 2016).
33. Schreiber GB, Glynn SA, Busch MP, Sharma UK, Wright DJ, Kleinman SH. Incidence rates of viral
infections among repeat donors: are frequent donors safer? Transfusion. 2001;41(6):730–5
(https://www.researchgate.net/publication/11939481_Incidence_rates_of_viral_infections_
among_repeat_donors_Are_frequent_donors_safer, accessed 20 December 2016).
188
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34. Zou S, Stramer SL, Dodd RY. Donor testing and risk: current prevalence, incidence, and
residual risk of transfusion-transmissible agents in US allogeneic donations. Transfus Med Rev.
2012;26(2):119–28 (abstract: https://www.ncbi.nlm.nih.gov/pubmed/21871776, accessed 20
December 2016).
35. O’Brien SF, Yi QL, Fan W, Scalia V, Fearon MA, Allain JP. Current incidence and residual risk of HIV,
HBV and HCV at Canadian Blood Services. Vox Sang. 2012;103(1):83–6 (abstract: https://www.
ncbi.nlm.nih.gov/pubmed/22289147, accessed 20 December 2016).
36. Bruhn R, Lelie N, Custer B, Busch M, Kleinman S. Prevalence of human immunodeficiency virus
RNA and antibody in first-time, lapsed, and repeat blood donations across five international
regions and relative efficacy of alternative screening scenarios. Transfusion. 2013;53(10 Pt 2):
2399–412 (abstract: https://www.ncbi.nlm.nih.gov/pubmed/23782054, accessed 20 December
2016).
37. Bruhn R, Lelie N, Busch M, Kleinman S. Relative efficacy of nucleic acid amplification testing and
serologic screening in preventing hepatitis C virus transmission risk in seven international regions.
Transfusion. 2015;55(6):1195–205 (abstract: https://www.ncbi.nlm.nih.gov/pubmed/25727549/,
accessed 20 December 2016).
38. Offergeld R, Ritter S, Hamouda O. HIV, HCV, HBV and syphilis surveillance among blood donors
in Germany 2008–2010. Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz.
2012;55(8):907–13 (article in German; English abstract: https://www.ncbi.nlm.nih.gov/
pubmed/22842883, accessed 20 December 2016).
39. Schreiber GB, Busch MP, Kleinman SH, Korelitz JJ. The risk of transfusion-transmitted
viral infections. N Engl J Med. 1996;334:1685–90 (http://www.nejm.org/doi/full/10.1056/
NEJM199606273342601#t=article, accessed 20 December 2016).
40. Glynn SA, Kleinman SH, Wright DJ, Busch MP. International application of the incidence
rate/window period model. Transfusion. 2002;42(8):966–72 (https://www.researchgate.
net/publication/11076616_NHLBI_Retrovirus_Epidemiology_Donor_Study_International_
application_of_the_incidence_ratewindow_period_model, accessed 20 December 2016).
41. Yoshikawa A, Gotanda Y, Minegishi K, Taira R, Hino S, Tadokoro K et al. Lengths of hepatitis B
viremia and antigenemia in blood donors: preliminary evidence of occult (hepatitis B surface
antigen-negative) infection in the acute stage. Transfusion. 2007;47(7):1162–71 (abstract:
https://www.ncbi.nlm.nih.gov/pubmed/17581150, accessed 20 December 2016).
42. Schreiber GB, Glynn SA, Satten GA, Kong F, Wright D, Busch MP et al. HIV seroconverting donors
delay their return: screening test implications. Transfusion. 2002;42(4):414–21 (https://www.
researchgate.net/publication/11299285_HIV_seroconverting_donors_delay_their_return_
screening_test_implications, accessed 20 December 2016).
43. an der Heiden M, Ritter S, Hamouda O, Offergeld R. Estimating the residual risk for HIV, HCV
and HBV in different types of platelet concentrates in Germany. Vox Sang. 2015;108(2):123–30
(abstract: https://www.ncbi.nlm.nih.gov/pubmed/25335096, accessed 20 December 2016).
44. Brambilla DJ, Busch MP, Dodd RY, Glynn SA, Kleinman SH. A comparison of methods for estimating
the incidence of human immunodeficiency virus infection in repeat blood donors. Transfusion.
2017;57(3pt2):823–31 (abstract: https://www.ncbi.nlm.nih.gov/pubmed/27910095, accessed
20 April 2017).
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Appendix 1
Evaluation of new blood-screening assays
Depending on the legal structure in a country, a regulatory body or the national
blood system itself may be responsible for decisions on the acceptability of new
blood-screening assays. It is recommended that previous assessments of quality
features of the assay performed by experienced regulatory authorities (for
example, United States FDA approval, European CE certification, and Australian
Therapeutic Goods Administration (TGA) or Health Canada marketing
authorizations) or by the WHO Prequalification Programme for IVDs should be
taken into account. Previous assessments by such stringent regulatory bodies will
have included the review of analytical and clinical performance data submitted
by the manufacturer, and of the manufacturer’s quality management system
and batch-to-batch consistency – and in the case of WHO prequalification, an
independent performance evaluation.
As a result, a country’s assessment of manufacturer documentation, with
a focus on the specific regional situation and needs, may be sufficient for assays
already approved elsewhere under stringent regulation.
If local regulation requires a performance evaluation of new assays (for
example, by a national reference laboratory) prior to their implementation, it is
recommended that the evaluation focuses on essential assay features through a
targeted performance evaluation.
Assessment of documents
Documents provided by the IVD manufacturer may be assessed, with a
WHO Technical Report Series, No. 1004, 2017
special focus placed on the specific regional situation and needs. Such a focus
may include assessing whether or not the stability studies performed by the
manufacturer cover the regional environmental conditions (for example, with
regard to temperature and humidity) or whether the Instructions for Use are
appropriate for the target users.
In addition, performance evaluation studies documented by the IVD
manufacturer may be reviewed to evaluate the extent of representation of
specimens reflecting the regional situation (for example, with regard to viral
genotypes or variants) or to assess potential interference with the test result by
other regionally more prevalent infections.
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Table A4A1.1
WHO reference preparations in the field of blood screening
Lyophilized
1000 IU/vial
HBsAg 3rd International Standard – NIBSC
Lyophilized
50 IU/mL
Dilutional panel – NIBSC
8.25; 2.06; 0.52; 0.13 IU/vial
1st International Reference HBV genotypes PEI
Panel HBV genotypes A–F, H
Lyophilized
No unitage
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Appendix 2
Examples for estimation of residual risks
Example 1: HCV screening by anti-HCV EIA
Centre A; observation period 01.06.2011–31.05.2012
49 660 repeat donors; 100 313 donations; 45 anti-HCV pos (EIA)
11 452 first-time donors; 11 452 donations; 89 anti-HCV pos (EIA)
Table A4.1 (see section 6.2 main text) – anti-HCV EIA: vDWP = 60 days =
0.164 years
Table A4.2 (see section 7 main text) – anti-HCV EIA: maximal virus
concentration: 10 8 IU HCV RNA/mL
plasma of vDWP donation
A. Residual risk (RR) per blood donation from repeat donors
number of repeat donors tested positive during one year
Incidence = × 100 000
total number of repeat donors in the year
45
= × 100 000
49 660
= 90.61 HCV infections per 100 000 donor years
RR per blood donation = vDWP × incidence
= 0.164 × 0.000 906 1 = 0.000 148 600
WHO Technical Report Series, No. 1004, 2017
A. Residual risk (RR) per blood donation from repeat donors (without adjustment
for transient HBsAg)
number of repeat donors tested positive during one year
Incidence = × 100 000
total number of repeat donors in the year
184
= × 100 000
49 660
= 370.52 HBV infections per 100 000 donor years
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Guidelines for the production, control and regulation of
snake antivenom immunoglobulins
Replacement of Annex 2 of WHO Technical Report Series, No. 964
1. Introduction 203
2. Purpose and scope 205
3. Terminology 205
4. The ethical use of animals 211
4.1 Ethical considerations for the use of venomous snakes in the production
of snake venoms 212
4.2 Ethical considerations for the use of large animals in the production
of hyperimmune plasma 212
4.3 Ethical considerations for the use of animals in preclinical testing of antivenoms 213
4.4 Development of alternative assays to replace murine lethality testing 214
4.5 Refinement of the preclinical assay protocols to reduce pain, harm and distress
to experimental animals 214
4.6 Main recommendations 215
5. General considerations 215
5.1 Historical background 215
5.2 The use of serum versus plasma as source material 216
5.3 Antivenom purification methods and product safety 216
5.4 Pharmacokinetics and pharmacodynamics of antivenoms 217
5.5 Need for national and regional reference venom preparations 217
6. Epidemiological background 218
6.1 Global burden of snake-bites 218
6.2 Main recommendations 219
7. Worldwide distribution of venomous snakes 220
7.1 Taxonomy of venomous snakes 220
7.2 Medically important venomous snakes 224
7.3 Minor venomous snake species 228
7.4 Sea snake venoms 229
7.5 Main recommendations 229
8. Antivenoms design: selection of snake venoms 232
8.1 Selection and preparation of representative venom mixtures 232
8.2 Manufacture of monospecific or polyspecific antivenoms 232
8.3 Main recommendations 234
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Abbreviations
ASV anti-snake venom
BVDV bovine viral diarrhoea virus
CK creatine kinase
CPD citrate phosphate dextrose solution
CTD Common Technical Document
ds-DNA double-stranded deoxyribonucleic acid
ds-RNA double-stranded ribonucleic acid
ED50 effective dose 50%
EIA enzyme immunoassay
ELISA enzyme-linked immunosorbent assay
EMCV encephalomyocarditis virus
FCA Freund’s complete adjuvant
FIA Freund’s incomplete adjuvant
GCP good clinical practice
GMP good manufacturing practice(s)
Hb haemoglobin
HPLC high-performance liquid chromatography
ICH International Conference on Harmonisation of Technical
Requirements for Registration of Pharmaceuticals for
Human Use
IgG immunoglobulin G
IgM immunoglobulin M
LD50 lethal dose 50%
MCD minimum coagulant dose
MDD minimum defibrinogenating dose
MHD minimum haemorrhagic dose
MHD50 MHD-median effective dose
MMD minimum myotoxic dose
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1. Introduction
Snake antivenom immunoglobulins (antivenoms) are the only therapeutic
products for the treatment of snake-bite envenoming. The lack of availability of
effective snake antivenom immunoglobulins to treat envenoming by medically
important venomous snakes encountered in various regions of the world has
become a critical health issue at global level. The crisis has reached its greatest
intensity in sub-Saharan Africa, but other regions, such as South and South-East
Asia, are also suffering from a lack of effective and affordable products.
The complexity of the production of efficient antivenoms, in particular the
importance of preparing appropriate snake venom mixtures for the production
of hyperimmune plasma (the source of antivenom immunoglobulins), the
decreasing number of producers, and the fragility of the production systems in
developing countries further jeopardize the availability of effective antivenoms
in Africa, Asia, the Middle East and South America. Most of the remaining
current producers are located in countries where the application of quality and
safety standards needs to be improved.
In October 2005, the WHO Expert Committee on Biological
Standardization recognized the extent of the problem and asked the WHO
Secretariat to support and strengthen world capacity to ensure the long-term
and sufficient supply of safe and efficient antivenoms. In March 2007, snake
antivenom immunoglobulins were included in the WHO Model List of Essential
Medicines (1), acknowledging their role in a primary health-care system.
WHO recognizes that urgent measures are needed to support the design
of immunizing snake venom mixtures that can be used to make appropriate
antivenoms for various geographical areas of the world. Sustainable availability
of effective and safe antivenom immunoglobulins must be ensured and
production systems for these effective treatments must be strengthened at global
level. Meaningful preclinical assessment of the neutralizing capacity of snake
antivenom immunoglobulins needs to be done before these products are used
in humans and medicines regulatory authorities should enforce the licensing of
these products in all countries, before they are used in the population.
The first edition of the WHO Guidelines for the production, control and
regulation of snake antivenom immunoglobulins was developed in response to
the above-mentioned needs and approved by the WHO Expert Committee on
Biological Standardization in October 2008. These Guidelines covered all the steps
involved in the production, control and regulation of venoms and antivenoms.
The Guidelines are supported by a WHO antivenoms database website1 that
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3. Terminology
The definitions given below apply to the terms as used in these WHO Guidelines.
These terms may have different meanings in other contexts.
Antivenom – also called antivenin or anti-snake venom (ASV): a purified
fraction of immunoglobulins or immunoglobulin fragments fractionated from
the plasma of animals that have been immunized against one or more snake
venoms.
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or a group of venoms, to react and neutralize the toxic effects of the venom of a
related species not included in the immunizing venom mixture.
Common Technical Document (CTD) format: a specific format for
product dossier preparation recommended by WHO and the International
Conference on Harmonisation of Technical Requirements for Registration of
Pharmaceuticals for Human Use (ICH).
Desiccation: a storage process where venoms are dehydrated under
vacuum in the presence of calcium salts or phosphoric acid.
Effectiveness: the effectiveness of an antivenom is a measure of its
ability to produce a clinically effective outcome when used to treat snake-bite
envenoming.
Efficacy: the efficacy of an antivenom is a measure of the in vivo or in
vitro neutralizing potency against a specific activity of a venom or venoms.
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(in mg/L or µg/mL) that clots either a solution of bovine fibrinogen (2.0 g/L) in
60 seconds at 37 °C (MCD-F) and/or a standard citrated solution of human
plasma (2.8 g/L fibrinogen) under the same conditions (MCD-P).
Minimum coagulant dose-F-effective dose (MCD-F100 ) and MCD‑P-
effective dose (MCD-P100 ): the minimum volume of antivenom or venom/
antivenom ratio, which completely prevents clotting induced by either one
MCD-F or MCD-P dose of venom.
Minimum defibrinogenating dose (MDD): the minimum amount of
venom that produces incoagulable blood in all mice tested within one hour
of intravenous injection.
Minimum defibrinogenating dose-effective dose (MDD100 ): the
minimum volume of antivenom or venom/antivenom ratio, at which the blood
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samples of all injected mice show clot formation after administration of one or
more MDD doses of venom.
Minimum haemorrhagic dose (MHD): The minimum amount of
venom (in µg) that when injected intradermally in mice causes a 10 mm
haemorrhagic lesion within a predefined time interval (for example, 2–3 hours).
Minimum haemorrhagic dose-median effective dose (MHD50 ): the
minimum volume of antivenom (in µL) that reduces the diameter of haemorrhagic
lesions by 50% compared to those induced in animals who receive a control
solution of venom/saline.
Minimum myotoxic dose (MMD): the minimum amount of venom that
produces a four-fold increase in serum or plasma creatine kinase (CK) activity
above that of control animals.
Minimum myotoxic dose-median effective dose (MMD50 ): the
minimum amount of antivenom (in µL or the venom/antivenom ratio) that
reduces the serum or plasma CK activity by 50% compared to those induced in
animals who receive a control solution of venom/saline.
Minimum necrotizing dose (MND): the minimum amount of venom
(in µg) that when injected intradermally in groups of lightly anaesthetized mice
results in a necrotic lesion 5 mm in diameter within 72 hours.
Minimum necrotizing dose-median effective dose (MND50 ): the
minimum amount of antivenom (in µL or the venom/antivenom ratio) that
reduces the diameter of necrotic lesions by 50% compared to those induced in
animals who receive a control solution of venom/saline.
Monospecific antivenom: antivenoms that are raised from venom of a
single species, and are limited in use to that species or to a few closely related
species (typically from the same genus) whose venoms show clinically effective
cross-neutralization with the antivenom. The term “monovalent” is often used
and has the same meaning.
Nanofilter: filters, most typically with effective pore sizes of 50 nm or
below, designed to remove viruses from protein solutions.
National regulatory authority (NRA): WHO terminology to refer to
national medicines regulatory authorities. Such authorities promulgate medicine
regulations and enforce them.
Plasma: the liquid portion remaining after separation of the cellular
elements from blood collected in a receptacle containing an anticoagulant, or
separated by continuous filtration or centrifugation of anticoagulated blood in
an apheresis procedure.
Plasmapheresis: procedure in which whole blood is removed from the
donor, the plasma is separated from the cellular elements by sedimentation,
filtration, or centrifugation, and at least the red blood cells are returned to
the donor.
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specific factual details of the GMP production and quality control manufacturing
activities that are undertaken at every site of operations linked to products that
a company produces.
Standard operating procedure (SOP): an authorized written procedure
giving instructions for performing operations not necessarily specific to a given
product or material (for example, equipment operation, maintenance and
cleaning; validation; cleaning of premises and environmental control; sampling
and inspection). Certain SOPs may be used to supplement product-specific
master and batch production documentation.
Toxin: a toxic substance, especially a peptide or protein, which is
produced by living cells or organisms and is capable of causing disease when
introduced into the body tissues. It is often also capable of inducing neutralizing
antibodies or antitoxins.
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of ethical use of animals, and WHO strongly recommends that every effort be
made to reduce pain, distress and discomfort to experimental animals – for
example, by the routine use of analgesia in mice used in these assays.
Analgesia should be considered, and evaluated to ensure that the humane benefits
of analgesia to the experimental animals do not invalidate the objectives of the
assay by altering relevant physiological processes (3). In particular, the use of
analgesia is recommended when working with venoms that induce tissue damage
(4). The establishment of humane end-points, instead of using survival/death as
the assay metric, is encouraged to reduce suffering and limit the duration of the
assays. The use of humane end-points also offers the opportunity to introduce
‘dose-staging’ into the experimental design (in which multiple doses are prepared
for the assays, one dose given and the next dose(s) selected based on the results of
giving the previous dose) to reduce the number of mice required for these assays.
All such efforts towards 3R require appropriate standardization and validation
within a quality assurance framework.
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5. General considerations
Snake antivenom immunoglobulins – antivenoms, antivenins, anti-snake-
bite serum and anti-snake venom (ASV) – are the only specific treatment for
envenoming by snake-bites. They are produced by the fractionation of plasma
that is usually obtained from large domestic animals hyperimmunized against
relevant venoms. Important but seldom used antivenoms may be prepared in
smaller animals. In general, when injected into an envenomed human patient,
an effective antivenom will neutralize toxins in any of the venoms used in its
production, and in some instances, will also neutralize venoms from closely
related species.
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6. Epidemiological background
The incidence of medically important snake-bites in different parts of the world
and the recognition of the species of greatest medical importance is fundamental
to the appropriate design of monospecific and polyspecific antivenoms in
countries and regions. Up-to-date epidemiological and herpetological information
is therefore highly relevant to antivenom manufacturers and regulators, especially
for the selection of the most appropriate venoms or venom mixtures to be used
in the production and quality control of antivenoms.
bite incidence within countries, the results of surveys of local areas cannot
be extrapolated to give total national values. Most of the available data suffer
from these deficiencies and, in general, should be regarded as underestimates
and approximations.
However, the true burden of national snake-bite morbidity and mortality
in three South Asian countries has recently been revealed by the results of three
well-designed community-based studies. In India, a direct estimate of 46 000
snake-bite deaths in 2005 was derived from the Million Death Study (30), in
Bangladesh there were an estimated 589 919 snake-bites resulting in 6,041 deaths
in 2009 (31), and in Sri Lanka in 2012–2013, 80 000 bites, 30 000 envenomings
and 400 deaths in one year (32). Published estimates of global burden, employing
highly controversial methodologies, suggest a range from a minimum of 421 000
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envenomings and 20 000 deaths up to as many as 2.5 million cases and more than
100 000 deaths each year (23, 33). In view of the recent data from South Asia,
these figures would seem to be underestimates. In addition, the number of people
left with permanent sequelae as a result of envenoming is likely to be higher
than the number of fatalities (21). As already identified, most of the estimated
burden of snake-bite is in sub-Saharan Africa, Central and South America and
South and South-East Asia.
The current literature on snake-bite epidemiology highlights the
inadequacy of the available data on this neglected tropical disease. There is
clearly a need to improve reporting and record-keeping of venomous bites in
health facilities, to support high-quality epidemiological studies of snake-bite in
different regions, and to improve the training of medical personnel. Wherever
possible, recording the species that caused the bite as well as death or injury
would greatly assist in documenting which species are of clinical significance in
individual countries. Making venomous bites notifiable and fully implementing
the use of the International Statistical Classification of Diseases and Related
Health Problems 10th Revision (34) in official death certification (for example,
T 63.0 snake venom) would further help to determine the burden of snake-bite
more accurately.
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Table A5.1
Genus-level name changes (1999–2016)
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Table A5.2
Changes resulting from new species descriptions or redefinitions (1999–2016)
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Table A5.3
Medically important venomous snakes: Africa and the Middle East
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Table A5.4
Medically important venomous snakes: Asia and Australasia
Central Asia
Elapidae: Naja oxiana; Viperidae: Echis carinatus; Gloydius halys; Macrovipera lebetina
East Asia
Elapidae: Bungarus multicinctus; Naja atra; Viperidae: Trimeresurus albolabris;
Daboia russelii; Deinagkistrodon acutus; Gloydius blomhoffii, Gloydius brevicaudus;
Protobothrops flavoviridis, Protobothrops mucrosquamatus; Trimeresurus stejnegeri
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South Asia
Elapidae: Bungarus caeruleus, Bungarus ceylonicus, Bungarus niger, Bungarus sindanus,
Bungarus walli; Naja kaouthia, Naja naja, Naja oxiana; Viperidae: Trimeresurus erythrurus;
Daboia russelii; Echis carinatus; Hypnale hypnale; Macrovipera lebetina
South-East Asia (excluding Indonesian West Papua)
Elapidae: Bungarus candidus, Bungarus magnimaculatus, Bungarus multicinctus,
Bungarus slowinskii; Naja atra, Naja kaouthia, Naja mandalayensis, Naja philippinensis,
Naja samarensis, Naja siamensis, Naja sputatrix, Naja sumatrana;
Viperidae: Calloselasma rhodostoma; Trimeresurus albolabris, Trimeresurus erythrurus,
Trimeresurus insularis; Daboia siamensis; Deinagkistrodon acutus
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Table A5.5
Medically important venomous snakes: Europe
Central Europe
Viperidae: Vipera ammodytes
Eastern Europe
Viperidae: Vipera berus
Western Europe
Viperidae: Vipera aspis, Vipera berus
Table A5.6
Medically important venomous snakes: the Americas
North America
Viperidae: Agkistrodon bilineatus, Agkistrodon contortrix, Agkistrodon piscivorus,
Agkistrodon taylori; Bothrops asper; Crotalus adamanteus, Crotalus atrox,
Crotalus horridus, Crotalus oreganus, Crotalus simus, Crotalus scutulatus,
Crotalus molossus, Crotalus viridis
Caribbean
Viperidae: Bothrops cf. atrox (Trinidad), Bothrops caribbaeus (St Lucia),
Bothrops lanceolatus (Martinique); Crotalus durissus (Aruba)
Central America
Viperidae: Bothrops asper; Crotalus simus
Pseudechis australis is common and widespread and causes numerous snake-bites; bites may be severe,
3
although this species has not caused a death in Australia since 1968.
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venoms from minor species within genera, or species whose bites are less
frequent than those of others in the same taxonomic groups (that is, genus,
subfamily or family).
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Fig. A5.1
Medically important North African and Middle Eastern venomous snakes:
(A) Egyptian cobra (Naja haje), (B) East Africa carpet viper (Echis pyramidum), (C) puff
adder (Bitis arietans), (D) Saharan horned viper (Cerastes cerastes) and (E) Levant viper
(Macrovipera lebetina)
A B
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D E
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Fig. A5.2
Medically important sub-Saharan African venomous snakes: (A) West African carpet
viper (Echis ocellatus), (B) Gaboon viper (Bitis gabonica), (C) Black mamba (Dendroaspis
polylepis), (D) Black-necked spitting cobra (Naja nigricollis), (E) Mozambique spitting
cobra (Naja mossambica)
A B
D E
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Main recommendations
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8.3
WHO Good Manufacturing Practices for Biological Products. WHO Technical Report Series, No. 996, 2016,
4
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Hide boxes should be designed to be slightly larger than the curled snake, with
an entrance/exit hole large enough to allow a recently fed snake easy access, plus
some simple closure device to lock the snake in the hide box. This will allow
removal of the snake from the cage without hazard to the keeper, making routine
cage maintenance simpler and safer. Hide boxes can be made from plastic, wood
or even cardboard (which is inexpensive and can be discarded and replaced
regularly). Permanent hide boxes should be readily cleanable or autoclavable. The
roof or side of the hide box should be removable, to allow easy and safe extraction
of the snake when required.
Cages should be thoroughly cleaned and disinfected when soiled (daily
if necessary). Faeces and uneaten or regurgitated food items should be removed
as soon as possible. To avoid misidentification of the snake, a microchip should
be implanted in the hypodermal layer of the snake’s posterior region and a label
bearing its individual data should be attached to the cage and transferred with
the snake when it is moved to another cage. Water should be freely available,
and for species from humid climates more frequent watering or misting may
be required, particularly when sloughing. Water should be changed regularly
and as soon as it becomes contaminated. Water treatment by ultraviolet (UV)
sterilization or acidification may be considered.
Tens of cages may be accommodated in the same “production room”,
provided that there is enough space for maintenance and venom extraction. This
room should be kept as clean as possible at all times, and thoroughly cleaned at
least weekly. Measures should be taken to minimize or eliminate contamination
or spread of diseases. The use of antiseptic hand washes, disposable over-clothing,
antiseptic foot wash trays at entry and exit points, and other measures should be
routine. The temperature and humidity of the snake room should be controlled
according to the climatic requirements of the particular snake species. Ventilation
should be ensured using fans, air-conditioning, or air renewing systems.
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can contain snakes of different species, provided that they require similar living
conditions (that is, temperature and humidity).
When kept under favourable housing and climatic conditions, and if left
undisturbed, snakes will reproduce in captivity (53). Animals should be mated
only with specimens from the same species, subspecies and local origin (54,
55). Sexing can be difficult, but is helped by the use of intra-cloacal probes. The
male and the female should be individually identified and separated soon after
copulation. The female should be kept under careful surveillance. Eggs from
oviparous snakes and neonates from viviparous snakes should be removed from
the female’s cage as soon as possible. Differences in the venom composition of
adult and juvenile snakes have been reported in some species (43, 48, 56–58), and
where this is known to occur or is suspected, the venom of a certain proportion
of juvenile snakes might be mixed with that of adults during the production of
venom batches.
The ideal frequency of feeding captive snakes depends on the species and
age of the snake, varying from twice per week to once per month. Snakes are
usually fed after venom extraction, ideally with dead mice or other appropriate
prey according to the snake species. Animals such as rats and mice that are raised
to feed snakes should be produced under appropriate quarantine standards in
facilities designed for this task. Humane euthanasia should be employed in the
killing of food animals, and ideally these food animals should be frozen for at
least 7 days before being thawed for use. Some snakes will only accept living prey,
but attempts should be made to wean them onto dead prey, and all local ethical
standards should be followed in the production and use of food animals. Snake-
eating species, such as kraits, coral snakes and king cobras, can be enticed to
take dead mice if the prey is first flavoured with snake tissue fluids, although any
such material should be frozen first for at least 7 days to kill parasites, before it is
thawed for use. Some coral species can be fed with fish strips (59). Living, dead or
regurgitated prey should not be left in the cage for more than a few hours. Force
feeding may be necessary for neonates and snakes that persistently refuse to feed.
Feeding time affords an opportunity to carefully check the snake for abnormal
behaviour, wounds, and possible infections and to give dietary supplements when
necessary. Individual feeding records are crucial. They should include details of
what, when and how prey was offered, when it was consumed and whether it
was regurgitated. The health of captive snakes can be estimated and recorded
by observing regular feeding and by measuring their weight and length. These
data are best stored on a computer system, using a “barcode” for each snake,
or, alternatively, using a reliable manual recording system, and constitute useful
records related to the venom batches produced. Venom extraction rooms should
be equipped with emergency eyewash stations and safety showers as is the case in
laboratories where there is a risk of chemical contact hazards.
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in the same room, because of the stress induced by the rats in the mice. The diets
required by young snakes may differ from those of adults (for instance, frogs and
tadpoles are preferred to rodents by some species), and facilities for producing
these food animals may also be required.
When possible, it is useful to have a small laboratory for performing
quality control on the venoms. All serpentariums need to be designed with
separate laboratories where venom can be processed after extraction and quality
control performed (see section 10). An area for repairing broken equipment
and for other miscellaneous purposes is also required. The administrative area
should be sufficiently large and adequately equipped with computer facilities so
that the traceability requirements needed for venom production can be met. The
whole venom production facility should be made secure against unauthorized
intrusion.
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from the glands, this may cause traumatic bruising to the animal and should be
avoided. The use of brief electrical impulses of moderate intensity to stimulate
venom secretion is not recommended. Any venom sample contaminated with
blood should be centrifuged. After venom extraction, the fangs are carefully
withdrawn from the collection vessel, while preventing damage to the mouth
and dentition and avoiding the snake impaling itself with its own fangs. Then, the
oral cavity should be sprayed with an antiseptic solution to avoid stomatitis. After
each venom extraction, all materials used in the process should be sterilized.
Peptides and proteins in venom are amphiphatic and will adhere to most
common surfaces including glass and plastic (60) resulting in the potential loss
of toxins from the venom used to produce hyperimmune plasma. The use of
polypropylene vessels and the addition of 1% bovine serum albumin can help
reduce such losses, but different peptides may have variable affinity for being
retained on vessel surfaces regardless of the approach taken to minimize loss.
Special procedures that avoid direct handling should be employed in
the case of burrowing asps (genus Atractaspis) because they cannot be held
safely in the way described above (61). For some species with small fangs and
small venom yields, the use of sterile pipette tips or capillary tubes which are
slipped over each fang one at a time, and pressure applied to the base of the
fang to stimulate venom release into the tube, is recommended. In the case of
colubrid snakes, special techniques are required, such as application of foam
rubber pads (from which venom is recovered in the laboratory) or pipette tips/
capillary tubes to the posteriorly placed fangs and the use of secretagogue drugs.
Similarly, some elapid snakes have small fangs and the pipette tip or capillary
tube technique is required to collect venom. At the time of venom extraction,
there is an opportunity to remove broken or diseased fangs and to examine the
snake for ectoparasites (for example, ticks and mites), wounds, dermatitis, areas
of adherent dead skin and retained spectacles over the eyes. The snake can be
treated with drugs and/or vitamins at the same time and, if necessary, can be
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force fed. When force fed with rodents, the rodent’s incisors must be cut out
so as not to cause any injury to the snake’s oesophagus. The process of venom
extraction is often combined with cage cleaning and disinfection and the feeding
of the snake. Avoiding trauma to the snake’s mouth and dentition is critical to
prevent infection and “mouth rot” and the venom extraction process should be
performed in accordance with clean practices.
Several snakes from the same group (same species and subspecies
collected at the same time in the same area) can have their venom collected
into the same venom collection vessel. The vessel should be kept in an ice bath
between individual extractions, and the venom aliquoted into labelled storage
tubes or vials and snap-frozen at −20 °C or colder within 1 hour. For venoms
with high proteolytic activity, the collected venom pool should be transferred
into a vial maintained at ultra-low temperature (−70 to −80 °C) or at least
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−20 °C, every 10–30 minutes, before continuing extractions from that group
of specimens. Another method is to transfer the collected venom into a vial
maintained in an ice bath. Refrigerated centrifugation of freshly collected
venom is recommended, for instance at 1000 g for 5 minutes (4 °C), to remove
cellular debris.
It is important to identify the vial into which the venom has been
collected or transferred for storage, with an appropriate reference number.
Primary indelible identification must be on the vial. This allows the identification
of all the snakes used during venom extraction, the name of the operator and any
other relevant information. To obtain large venom batches for the preparation
of antivenom, especially from species with low yields, one approach is to use
the same vial over several months for extractions performed with the same
specimens, providing the cold chain is never broken. Pools of venom require
unique batch numbers, and the individual venom extractions contributing to the
pool must be traceable. When a pool is sufficient in volume, the venom should
be either freeze- or vacuum-dried and kept in the dark at a low temperature
(either −20 °C or 4 °C) in a well-sealed flask, precisely identified with a number,
up to the time of delivery. Some producers use an alternative system, keeping
dried venom at 20–25 °C in a desiccator. Regardless of the method used, the
procedures for drying venom should be well established, documented, validated
and incorporate appropriate quality control steps (for example, periodic
determination of residual moisture against established standards). The potency
of venom stored for considerable periods of time must be tested at least annually
to ensure that no degradation or loss of activity has occurred (see section 10),
and if a loss of potency is observed the batch must be replaced.
The equipment used for storage of frozen venom (freezers) and for
venom drying, should be cleaned using established procedures, and the cleaning
documented, in order to minimize cross-contamination. Likewise, equipment
requiring calibration, such as freezers, balances and freeze-driers, should be
calibrated according to a defined schedule.
impairs manual dexterity and sense of touch, but the use of nitrile gloves is
advisable to prevent cross-contamination. Puncture-resistant gloves should be
mandatory as protective equipment for assistants helping to restrain or handle
snakes during procedures such as venom extraction.
When lyophilized or desiccated venom is being handled, the safety of
operators is paramount, since dried venom can easily be aerosolized and affect
people through skin breaks, eyes or mucous membranes, or by sensitizing them
to the venom (68). Appropriate gowning is necessary when handling dried or
liquid venom, to prevent contact of the venom with skin or mucous membranes.
It is highly recommended that a biological safety cabinet (for example, Class II,
B2), be used while handling lyophilized or desiccated venom.
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supply of the correct antivenom can accompany the victim to hospital. Hospital
staff should be warned in advance by telephone of the arrival of the casualty and
informed about the species responsible and any background medical problems
and relevant medical history, such as past reactions to antivenom or other equine
sera (for example, anti-tetanus serum), and known allergies.
the place of capture recorded. Genetic samples (for example, tissue and blood)
should be routinely collected from all specimens for DNA analysis if questions
arise regarding the validity of the identification of specimens. Photographs of
individual specimens may also have value.
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Fig. A5.3
General manufacturing process of antivenoms
Veterinary surveillance
Preparation of immunizing
doses of venoms
Immunization programme
for each animal
for fractionation
Fractionation of plasma
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tested for.
The immune response against venom components should, when
feasible, be followed throughout the immunization schedule, in order to detect
when animals reach an acceptable antivenom titre. However, the monitoring
of the immune response can be done on a pool of sera from various animals.
This response may be followed by in vivo potency assays of neutralization of
lethality or by in vitro tests, such as enzyme immunoassays (EIAs) (provided
that a correlation has been demonstrated between these tests and the in vivo
potency tests).
Whenever an animal develops any manifestation of sickness, it must be
temporarily withdrawn from the immunization programme to allow it to receive
appropriate veterinary examination and treatment. If the disease is controlled,
the animal may return to the immunization programme after a suitable length
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In some areas, legislation stipulates that animals used for production of plasma cannot be treated with
6
penicillin or streptomycin.
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care should be taken when manipulating them. When taken out of the refrigerator
or freezer, the venom should be allowed to warm up to room temperature before
the bottle is opened. Otherwise, condensation may occur causing inaccuracy in
weighing and, more seriously, proteolytic degradation of the venom proteins by
venom enzymes. Venom should be dissolved in distilled water or buffer, but care
should be taken not to shake the solution too vigorously since excessive foaming
may cause protein denaturation.
The solvents used to dissolve venoms should be sterile and should be
used before the established expiry dates. A stock solution of each venom should
be prepared separately, rather than being mixed with other venoms. This is to
allow flexibility of dosage and to avoid proteolytic degradation by one venom
component of other venom proteins. Venom solutions can be sterile filtered
(where this is known not to affect the potency of the preparation), aliquoted
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mineral oil and an emulsifier. FCA, which contains mineral oil, an emulsifier
and inactivated Mycobacterium tuberculosis, has been shown in experimental
animals to be one of the most potent adjuvants known. However, horses are quite
sensitive to FCA which tends to cause granuloma formation. For this reason,
some producers prefer to use other adjuvants. It is recommended that when
using FCA and FIA, they be utilized only at the beginning of the immunization
schedule, and not during the rest of the immunization, nor during booster
injections of venom; this significantly reduces the formation of granulomas
in horses.
It has been noted that the granuloma caused by FCA is caused by the
injection of a large volume (5–10 mL) of the emulsified immunogen at one or
two sites. The large granuloma formed usually ruptures, resulting in a large
infected wound. If the emulsified immunogen is injected subcutaneously in small
volumes (50–200 µL/site) at multiple sites, granuloma formation may be avoided.
Manufacturers are also encouraged to adopt an innovative approach with regard
to adjuvants used for antivenom production, and should strive to replace FCA
and FIA with new compounds of low toxicity and high adjuvant effect. The
advances in the vaccine field concerning new adjuvants should be transferred to
the antivenom field; for example, the use of microbial-derived products of low
toxicity or of Toll-like receptor 4 (TLR4) ligand-based adjuvants (80).
Fig. A5.4
Tuberculin syringes are filled with immunogen suspension and used for the
subcutaneous injection of the horse
Box A5.1
Example of preparation of venom immunogen in FCA, FIA and aluminium salts
Since FCA can cause severe irritation, precautions should be taken to avoid contact with
the eyes, and protective eyewear and gloves are recommended. The vial containing
FCA is shaken to disperse the insoluble Mycobacterium tuberculosis. The venom
solution is mixed in a stainless steel container with an equal volume of FCA at 4 ºC.
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Fig. A5.5
Recommended areas of immunization in horses
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Box A5.2
Example of immunization of horses using FCA, FIA and aluminium salts
The primary immunization could be done with venom(s) mixed with FCA as described
in Box A5.1. The initial dose of each venom could be as low as 1–4 mg/horse with a
total combined volume of injection of about 2 ml. The immunogen is filled in several
1 ml tuberculin syringes with 21G needles as described in Box A5.1 and Fig. A4.5.
Subcutaneous injections of 100–200 µL of immunogen are made at each site, up to
as many as 8–12 sites, although some producers may use only 3–4 injection sites.
The neck of the horse, supplied with extensive lymphatic vessels and large lymph
nodes, is a preferred area for immunization. If inoculation is made on the lateral
sides of the neck, the animal tends to rub itself causing skin blisters. Thus, injections
should be made to the upper (dorsal) part of the neck, close to the mane. About 4–6
injections can be made at each side of the neck. If injection in the rump is possible,
1–2 injections can be made in the area between the outer hip bone and the top of
the thigh bone. The scratching of injected sites by animals can be partially alleviated
by massaging the injection site after venom injection to disperse the dose material.
Immunization using FCA is usually done only once; in most cases, repeated use of this
adjuvant can cause serious reactions which affect the horse’s health. After 2 weeks,
the horses should receive a booster injection with the same venom(s) well emulsified
in FIA. Similar volumes and areas of injection to those described above can be used.
Subsequent booster immunizations at 2-week intervals can be administered, with
higher doses (5–10 mg) of venom(s) in saline or mixed with aluminium salts or any other
suitable adjuvant. In this case, subcutaneous injections of 1 ml of immunogen at each
site in a total of 4 sites are recommended. Blood (10–20 ml) should be drawn before
each immunization. Serum or plasma is prepared and EIA titres and/or lethality potency
are determined. When the EIA titres reach a plateau, usually about 8–10 weeks after the
primary immunization, an in vivo potency assay may be performed to confirm that the
horse could be bled. After bleeding for antivenom production, the horses are allowed
4–8 weeks rest, depending on their physical condition. After the rest period, a new round
of immunization can be performed as described above, but without the use of FCA.
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14.4.1.2 Storage
The bags or bottles in which the whole blood has been collected should be
appropriately cleaned and sanitized on their external surfaces. They should
be put into a refrigerated room (2–8 °C) for the plasma and blood cell separation
procedure. They should be stored for no more than 24 hours before the
reinfusion of the red cells, unless CPD is used. In this case, blood cells may be
stored for up to 72 hours. Alternatively, aseptically collected blood can be stored
for a maximum of 7 hours at 20–25 °C to allow for sedimentation. Under such
circumstances, great care should be taken to avoid bacterial contamination.
should take place within 24 hours after blood collection (or 72 hours if CPD
anticoagulant is used), and after being suspended in sterile saline solution at
room temperature for 1 hour (or 32–37 °C for less than 1 hour) prior to infusion.
This procedure in which whole blood is collected and red blood cells are
re‑infused into the animal is commonly referred to as “manual apheresis”.
during reinfusion of the red blood cells to the donor. Plasma from automatic
apheresis tends to be less contaminated by blood cells (red blood cells, leukocytes
and platelets). In the experience of some laboratories the plasma is easier to
fractionate, as the filtration steps, in particular, are more readily performed,
resulting in higher yields.
In such procedures, whole blood is collected from the animal, mixed
with anticoagulant, and passed through an automated cell separator. The
plasma is separated from the cellular components of the blood, which are
returned to the animal in a series of collection/separation and return cycles.
The plasma is separated from the red blood cells by centrifugation or filtration,
or a combination of the two. The operational parameters of the plasmapheresis
equipment are provided by the manufacturers of the equipment. In general, the
anticoagulant is delivered at a rate yielding a specified ratio of anticoagulant to
blood. The anticoagulant solutions used include AB16 (35.6 g sodium citrate,
12.6 g citric acid monohydrate, 51.0 g glucose monohydrate per litre using water
for injection) and anticoagulant citrate dextrose formula A (ACDA) (22.0 g
sodium citrate, 8.0 g citric acid, 24.5 g dextrose monohydrate, per litre using
water for injection). The number of collection/separation and return cycles
for each donor animal depends on the total volume of plasma that is to be
harvested. For horses, the average volume of plasma collected may be about 6
litres per session. The number of cycles ranges from 10 to 20 depending upon
the haematocrit of the horses. The collection process lasts for 1–4 hours. The
apheresis equipment and apheresis procedures should be validated, maintained
and serviced. Machine plasmapheresis can take several hours and animals can
be fed during the operation.
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avoided and the cold chain is maintained. To avoid the risk of contamination,
it is recommended that individual or pooled plasma is not stored for too long
before fractionation, that is, the plasma should be fractionated as soon as possible
after pooling.
If plasma is stored for prolonged periods, the storage time and conditions
should be validated to ensure that there is no detrimental impact on the quality
of the plasma material, on the fractionation process, or on the quality, efficacy
and stability of the antivenoms.
Manufacturers of human plasma have found that plasma can be stored
frozen at −20 °C or colder for 2 years without addition of a preservative, and
with no observed detrimental effects on the fractionated plasma products.
14.5 Pooling
Plasma from individual animals should be pooled into sterile and sanitized
containers before fractionation. For traceability purposes each plasma pool
should be identified with a unique number. The number of plasma units collected
from individual animals and used in the pool should be recorded. Before the
large pool of plasma is prepared, it is recommended to prepare a small-volume
pool and to test it for microbial contamination. If there is no contamination,
the large pool can be prepared. If microbial contamination is detected, the
plasma from the individual animals should be checked, and the contaminated
plasma should be discarded to ensure that the pool is prepared with plasma free
of microbial contamination. It may also be advisable to test small pools using
a cytotoxicity assay, which can reveal the presence of unanticipated viruses
or toxins (for example, following the US Code of Federal Regulations, 9 CFR
113.53 “Requirements for ingredients of animal origin used for production
of biologics”).8
Such pooling should be performed in an environment suitable to prevent
WHO Technical Report Series, No. 1004, 2017
microbial contamination, for example, classified areas (class D (83)) and pools
should be adequately identified. The room should be designed to allow for
appropriate cleaning and sanitization of all surfaces. Individual or pooled plasma
should be stored at 2–8 °C in a room dedicated for this purpose. To ensure the
prevention of microbial contamination of plasma, follow the recommendation
given in section 14.4.2.2.
https://www.gpo.gov/fdsys/search/pagedetails.action?packageId=CFR-2005-title9-vol1&granuleId=CFR-
8
2005-title9-vol1-sec113-53&bread=true&collectionCode=CFR&browsePath=Title+9%2FChapter+I%2F
Subchapter+E%2FPart+113%2FSubjgrp%2FSection+113.53&collapse=true&fromBrowse=true, accessed
24 April 2017.
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results. Each manufacturer should adjust the pepsin concentration to achieve the
required enzymatic activity.
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Fig. A5.6
Example of a fractionation process in which intact IgG is prepared by caprylic acid
precipitation of non-immunoglobulin proteins
Fractionation of plasma for purification of IgG
Hyperimmune plasma
Filtration or centrifugation
Discard the precipitate
Filtrate or supernatant
Tangential diafiltration
and concentration
Bulk preparation
Dispensing in final
container
Final product
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Fig. A5.7
Example of a fractionation process in which F(abʹ)2 fragments are prepared by pepsin
digestion and ammonium sulfate precipitation
Fractionation of plasma for purification of F(abʹ)2 fragments
Hyperimmune plasma
Filtrate or supernatant
Filtrate or supernatant
F(abʹ)2 paste
WHO Technical Report Series, No. 1004, 2017
Solubilization of precipitate
Tangential flow diafiltration and
concentration
Bulk preparation
Dispensing in final
container
Final product
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Fig. A5.8
Example of a fractionation process in which F(abʹ)2 fragments are prepared by pepsin
digestion and caprylic acid precipitation
Fractionation of plasma for purification of F(abʹ)2 fragments
Hyperimmune plasma
Filtration or centrifugation
Discard the precipitate
Filtrate or supernatant
Adjust pH to 7.0
Bulk preparation
Dispensing in final
container
Final product
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Fig. A5.9
Example of a fractionation process in which Fab fragments are prepared by papain
digestion and ammonium sulfate precipitation
Fractionation of plasma for purification of Fab fragments
Hyperimmune plasma
Filtration or centrifugation
Discard the supernatant
IgG-rich precipitate
Solubilization of precipitate in
buffered saline solution
Fab solution
Anion-exchange chromatography
and concentration
Bulk preparation
Dispensing in final
container
Final product
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15.2.5 Formulation
During formulation of antivenoms after diafiltration steps one should consider
the addition of salts to adjust the osmolality, addition of preservatives, other
excipients, if needed for protein stability, and the adjustment of pH.
In general, antivenoms are formulated at neutral pH (pH 7.0 ± 0.5)
although some manufacturers are exploring the feasibility of formulation at more
acidic pHs to improve stability and/or to reduce aggregate formation.
Formulation at a pH higher than 7.5 may not be recommended, since the
stability of immunoglobulins and their fragments at alkaline pH may be poor,
and the formation of aggregates may be favoured.
also be validated.
due to heat. For vials, insertion of rubber stoppers should be done inside this
clean dispensing area. The quality of the rubber stoppers should be such as to
guarantee inertness and to prevent leaching. Thereafter, aluminium seals should
be placed on each vial in a clean area outside the class A area. Ampoules or vials
containing the final product should then be properly identified and stored in
a quarantine area maintained under proper storage conditions. Samples of the
antivenoms should be sent to the quality control laboratory for analysis.
When an antivenom complies with all the quality control tests established
for the final product, it should be properly labelled and identified.
■■ The vial or ampoule should be labelled with, at least, the following
information:
(a) name of the product and of the producer;
(b) animal species used to produce the antivenom;
(c) batch number;
(d) pharmaceutical presentation (liquid or freeze-dried);
(e) volume content;
(f ) administration route;
(g) specificity – venoms neutralized by the antivenom, including
both the common and the scientific name of the snake(s);9
(h) neutralizing potency;
( i ) storage conditions; and
( j ) expiry date.
Additional information may be requested by NRAs.
■■ The package, which is usually a cardboard box, in which the vials
or ampoules are packed, should include the same information as is
given on the primary container.
■■ The package insert should include all the information relating to the
product, as established by NRAs, including:
(a) the neutralizing potency;
(b) the recommended dosage;
(c) reconstitution procedure, if lyophilized;
(d) the mode of administration (for example, the dilution of
antivenom in a carrier fluid such as saline);
Special care should be taken to consider potential changes in snake species taxonomy.
9
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15.2.9 Freeze-drying
Antivenoms are available either as liquid or as freeze-dried preparations. Freeze-
dried antivenoms, which may usually be stored at a temperature not exceeding
25 °C, are generally distributed to markets where the cold chain cannot be
guaranteed, such as in many tropical regions of the world. The absence of guarantee
of a cold chain during distribution highlights the need for manufacturers to
demonstrate the stability of the antivenoms under the high temperatures found
in tropical climates.
Freeze-drying is a critical operation. Careful attention should be paid to
the rate of freezing as well as to the protocol used for the primary and secondary
drying cycles (102). The details of the freeze-drying protocols are product-
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neutralized toxins reach the circulation. This gives rise to the well-known
phenomenon of recurrent envenoming, that is, the reappearance of signs and
symptoms of envenoming at later time intervals after the initial control of
envenoming. This situation demands repeated administration of antivenom to
maintain therapeutic levels of Fab in the circulation (110). Therefore, in such
envenomings, antivenoms made of IgG or F(abʹ)2 may be more appropriate
because of their longer elimination half-lives. Moreover, it has been proposed
that formation of venom–antivenom complexes in the circulation results in the
redistribution of venom components from the extravascular space to the blood
compartment, where they are bound and neutralized by circulating antivenom,
provided that the dose of antivenom is sufficient (111, 112). Consequently, the
maintenance of a high concentration of specific antivenom antibodies in the
circulation for many hours is required for complete neutralization of toxins
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reaching the bloodstream during both early and late phases of envenoming
(redistribution of toxins) present in the extravascular space. In conclusion, IgG
and F(abʹ)2 antivenoms have a pharmacokinetic profile that makes them more
effective in many types of snake-bite envenoming.
comprising at least one viral inactivation step) should provide a satisfactory level
of viral safety. However, it should be kept in mind that non-enveloped viruses
are more difficult to inactivate or remove than lipid-enveloped viruses.
Table A5.7
Viruses identified in horses (86, 113)
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encephalitis
virus
(116, 117)
Venezuelan Togaviridae 40–70 ss-RNA Yes Yes
equine
encephalitis
virus
Vesicular Rhabdoviridae 50–80 ss-RNA Yes Yes
stomatitis
virus
West Nile Flaviviridae 40–70 ss-RNA Yes Yes
virus
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Table A5.8
Viruses identified in sheep and goats (86)
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inactivation step, it is highly recommended that the kinetics of the virus kill
be evaluated. Such inactivation kinetics of the infectivity provide an important
indication of the virucidal potential of the step and enables comparison of the
data obtained to those from published studies.
Typically, a viral reduction of 4 logs or more is considered to represent
an effective and reliable viral safety step.
Establishing the relative insensitivity of a manufacturing step to changes
or deviations in process conditions is also important in evaluating its robustness,
in addition to adding to the level of understanding of its contribution to the
overall viral safety of the preparation. This can be achieved by validating the
same step using a range of conditions deviating from those used in production
(such as an upper pH limit applied to a pepsin digestion or to a caprylic acid
precipitation step).
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Table A5.9
Examples of laboratory model viruses that can be used for validation studies of horse-derived antivenoms
Table A5.10
Comparison of conditions for caprylic acid treatment used for human immunoglobulin
preparations and antivenoms (113)
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Virus inactivation studies have been carried out on an F(abʹ)2 fraction obtained
from pepsin-digested plasma subjected to ammonium sulfate precipitation. The
F(abʹ)2 fraction was subjected to precipitation by drop-wise addition of caprylic
acid to 0.5% (final concentration) and the mixture was maintained under
vigorous stirring for 1 hour at 18 °C. Rapid and complete reduction of BVDV,
pseudorabies virus and VSV (> 6.6 log 10 , > 6.6 log 10 , and > 7.0 log 10 , respectively)
was observed. No significant reduction (0.7 log 10 ) of the non-enveloped EMCV
(126) was observed.
In another process used to prepare equine immunoglobulins, serum
is thawed at 4 °C, subjected to heating at 56 °C for 90 minutes, brought to
20 ± 5 °C, adjusted to pH 5.5 and subjected to 5% caprylic acid treatment
for 1 hour. This process led to fast reduction of infectivity of > 4.32 and
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> 4.65 log 10 for pseudorabies virus and BVDV, respectively. The caprylic acid
step was confirmed to have only limited impact on the infectivity of EMCV
and minute virus of mice (MVM) non-lipid-enveloped viruses (128). Data
suggest that significant reduction in the infectivity of lipid-enveloped viruses
can be obtained during caprylic acid treatment of antivenoms. The reduction of
viral infectivity may result from both viral inactivation and partitioning during
the precipitation step. No significant inactivation of non-enveloped viruses
is expected.
Table A5.11
Typical conditions for acid pH treatment of human IgG preparations and equine
antivenoms (113)
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as less robust than those resulting from dedicated viral inactivation or removal
steps (84).
16.4.4.2 Nanofiltration
Nanofiltration is a technique of filtration specifically designed to remove viruses,
based on size, while permitting flow-through of the desired protein (131).
Effective virus removal requires, in principle, that the pore size of the filter be
smaller than the effective diameter of the virus particles.
However, the blood of sheep with experimental BSE or natural scrapie can be
infectious and, because scrapie and BSE prion agents behave similarly in sheep
and goats, the use of the blood of small ruminants in preparing biologicals should
either be avoided or the animals should be selected very carefully from sources
known to be free of TSEs. The findings of disease-associated proteins in muscle
tissue of sheep with scrapie and the recognition of BSE itself in a goat, reinforce
the need for manufacturers of biologicals, including antivenoms, to maintain the
precautionary safety measures recommended in the WHO guidelines on TSE
tissue infectivity (77).
According to these recommendations, the use of tissues or body fluids
of ruminant origin should be avoided in the preparation of biological and
pharmaceutical products. When sheep-derived materials must be used, they
should therefore be obtained from sources assessed to have negligible risk
from the infectious agent of scrapie. Documented surveillance records should
be available. The feed of animals used for production of hyperimmune plasma
should be free of ruminant-derived material.
The infectious agent is thought to be a misfolded, abnormal, prion protein
(PrPTSE). It is not yet known whether manufacturing processes used to produce
antivenoms from sheep plasma include steps that can contribute to the removal
of PrPTSE. Experimental prion clearance studies, based on spiking experiments,
can be performed to assess the capacity of the process to remove prions. However,
there is still uncertainty about the validity of such experimental studies since the
biochemical features of PrPTSE in blood and plasma are not known.
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on the final bottled product if the processing after the bulk preparation has been
validated and shown not to have any impact. Quality control assessment of the
final antivenom product includes the tests described below.
of: venom lethality and antivenom efficacy, which enable internal monitoring
and external, independent auditing of antivenom efficacy – thereby preventing
the distribution of ineffective antivenom.
Consistent use of outbred strains of mice, of a defined weight range (for
example, 18–20 g) that receive a defined challenge dose, is recommended for all
the assays. Some producers use other test animals, such as guinea-pigs. While
weights will clearly vary between animal species, a series of principles, specified
for mice, will still apply to these alternative test animals. It should be borne in
mind that there are variations in the susceptibility of different strains of mice
to the lethal effect of venoms.
The venom-neutralizing potency tests are used for quality control and
preclinical assessment, so protocol details are described in section 19, while
ethical issues are discussed in section 4.
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17.1.5 Osmolality
Osmolality is used to measure the tonicity of the antivenom solution, and should
be at least 240 mOsmol/kg. Determination of osmolality is also an indirect
means to determine the quantity of salts or excipients added for formulating
the batch.
17.1.14 Determination of pH
The pH of antivenom should be determined using a potentiometer.
18.2 Storage
Antivenoms should be stored at a temperature within the range that assures
stability, as found by stability tests. This is particularly critical for liquid
formulations, which usually require storage at between 2 and 8 °C. Therefore,
deviations from this temperature range, due to interruptions in the cold chain
during transportation or storage, are likely to result in product deterioration. The
design of adequate cold chain programmes, as part of the public health systems
in every country, is critical, and national protocols should be developed. The
distribution policies for national vaccination programmes can be adopted for
the transportation and storage of antivenoms. The stability of liquid preparations
at temperatures higher than 2–8 °C should be evaluated and, if needed, new
formulations allowing such storage conditions should be developed.
18.3 Distribution
Adequate distribution of antivenoms is a matter of great concern in many
regions of the world. Since most of the antivenoms available are liquid
preparations, the maintenance of an adequate cold chain must be guaranteed,
despite the difficulties to be encountered in rural areas of some developing
countries. National and regional health authorities should develop distribution
strategies to ensure that antivenoms are allocated to the areas where they are
needed or use the distribution channels in place for other national primary
health-care programmes. Both the specificity of the antivenom and the number
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These tests determine the capability of an antivenom to neutralize the lethal effect
of the snake venom(s). It is first necessary to determine the lethal potency of
the venom using the LD50 assay. The exact volume of antivenom, or the venom/
antivenom ratio, required to neutralize venom lethality can then be determined
using the antivenom effective dose (ED50 ) assay.
Preclinical testing of antivenom is required:
■■ for routine quality control of efficacy of each newly manufactured
batch of an existing antivenom;
■■ to test the ability of a new antivenom to neutralize the lethal effects
of venoms from snakes from the country or region where it is going
to be introduced;
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mixtures before use is not recommended because residual venom toxicity may
remain in the immuno-precipitate.
After injection, deaths are recorded at 24 hours (intravenous test) or at
48 hours (intraperitoneal test) and the results analysed using Probit analysis,
Spearman-Karber or alternative procedures (such as non-parametric methods).
One antivenom ED50 dose is defined as the amount of antivenom, or the venom/
antivenom ratio, resulting in the survival of 50% of mice injected with a mixture
of antivenom and a lethal quantity of venom.
The ED50 result can be expressed in various ways:
■■ mg of venom neutralized by mL of antivenom;
■■ µL antivenom required to neutralize the “challenge dose” of venom
used;
■■ µL of antivenom required to neutralize 1 mg of venom.
The practice of defining the ED50 by the number of murine LD50 s of
venom neutralized per mL of antivenom is inaccurate and has little clinical
usefulness. Since LD50 values for the same venom may vary from one
manufacturer to another, this representation of ED50 should be avoided in
favour of one of the approaches listed above.
19.3.1
Many venoms, especially those of vipers, exert powerful local and systemic
haemorrhagic activity effected primarily by snake venom zinc-dependent
metalloproteinases. These enzymes damage the basement membrane that
surrounds the endothelial cells of capillaries resulting in bleeding into the
tissues. Bleeding into the brain and other major organs is considered to be the
major lethal effect of envenoming by many viperid species (144). The minimum
haemorrhagic dose of a venom (MHD) quantifies this venom-induced pathology,
and is defined as the amount of venom (in µg dry weight) which, when injected
intradermally, induces in mice a 10 mm haemorrhagic lesion after a predefined
time interval, usually 2–3 hours, after injection (145, 146).
The venom MHD test is carried out by preparing aliquots of 50 µL of
physiological saline solution containing a range of venom doses. Mice (18–20 g
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body weight; five mice per group) are held securely and the hair surrounding
the injection site is shaved. Venom solutions (50 µL) are injected intradermally
into the shaved skin of lightly anaesthetized mice. After a defined time interval
(usually 2–3 hours), mice are killed using an approved humane procedure, the
area of the injected skin is removed, and the size of the haemorrhagic lesion
in the inner side of the skin is measured using calipers in two directions with
background illumination. Care should be taken not to stretch the skin. The mean
diameter of the haemorrhagic lesion is calculated for each venom dose and the
MHD estimated by plotting mean lesion diameter against venom dose and
reading off the dose corresponding to a 10 mm diameter (145, 146).
The assay measuring the efficacy of antivenom to neutralize venom-
induced haemorrhage is termed the MHD-median effective dose (MHD50 ),
and is defined as the volume of antivenom, in microlitres, or the venom/
antivenom ratio, which reduces the diameter of haemorrhagic lesions by 50%
when compared with the diameter of the lesion in animals injected with the
control venom/saline mixture (146). A “challenge dose” of venom is selected –
between one and five venom MHDs have been used as the challenge dose by
different laboratories. The test is carried out as above, using five mice per group.
Mixtures of a fixed amount of venom and various dilutions of antivenom are
prepared so that the challenge dose of venom is contained in 50 µL. Controls
must include venom solutions incubated with physiological saline solution
alone. Mixtures are incubated at 37 °C for 30 minutes, and aliquots of 50 µL
are injected intradermally in lightly anaesthetized mice. The diameter of
haemorrhagic lesions is quantified as described above, and the neutralizing
ability of antivenom is expressed as the MHD50 .
mice (18–20 g body weight), results in a necrotic lesion of 5 mm diameter 3 days
later. The method used is the same as that for the MHD, except that the skin is
examined 3 days after the intradermal injection of the venom (145).
The assay measuring the ability of an antivenom to neutralize venom-
induced dermonecrosis is termed the MND-median effective dose (MND50 ), and
is defined as the volume of antivenom, in microlitres or the venom/antivenom
ratio, which reduces the diameter of necrotic lesions by 50% when compared
with the diameter of the lesion in mice injected with the control venom/saline
mixture. A challenge dose of venom is selected, usually between one and two
MNDs. The test is carried out as above, using five mice per group. Mixtures of a
fixed concentration of venom and various dilutions of antivenom are prepared
so that the venom challenge dose is contained in 50 µL. Controls include venom
solutions incubated with physiological saline solution alone. Mixtures are
incubated at 37 °C for 30 minutes, and aliquots of 50 µL are injected intradermally
in lightly anaesthetized mice (151, 152). The diameter of dermonecrotic lesions
is quantified 3 days post-injection, as described above, and the neutralization by
antivenom, expressed as the MND50 .
11
See http://www.therqa.com/committees-working-parties/good-clinical-practice/regulations-and-
guidelines/, accessed 23 April 2017.
12
http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500002874.
pdf, accessed 23 April 2017.
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https://clinicaltrials.gov/ct2/manage-recs/how-register; http://www.isrctn.com/;
13
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about whether the benefit (degree of clinical improvement) from the antivenom
outweighs the risk (potential incidence and severity of adverse events). Depending
on the speed of evolution of envenoming, immediate treatment might be
compared to delayed antivenom treatment. A new antivenom with demonstrable
preclinical potency (see above) can be compared with an established product, or
two markedly different initial doses or regimens of the same antivenom can be
compared. In RCTs, non-inferiority, rather than superiority of a new antivenom
or regimen, compared to an existing treatment, requires smaller numbers of trial
participants to achieve acceptable power (94). Basic requirements for any clinical
antivenom RCT are that the participants should be reasonably homogeneous as
far as the species of snake responsible and their pretreatment characteristics (for
example, interval between bite and treatment) are concerned, and that objective
clinical end-points should be selected to judge effectiveness, and measure rates
of adverse events.
and are particularly useful in defining safety or when RCTs are unethical or
impracticable. In these, the proportion of patients with good clinical outcomes
(for example, restoration of blood coagulability or failure to develop local wound
necrosis) can be observed with different, escalating or de-escalating doses of
antivenom. However, the many weaknesses of observational studies must always
be borne in mind.
As part of the design of the study, it is important to determine the
minimum number of patients required to establish meaningful results by using
sample size calculations (168). Results may sometimes be compared to those of
previous studies (historical controls) to determine how the effectiveness or safety
of a newly introduced antivenom compares with previously used antivenoms
(169). However, such comparisons are susceptible to many kinds of confounding
variables and are potentially unreliable. Subsequently, the minimum dose that
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Major changes in the design, manufacturing process, or source of venoms used for production of
15
antivenom may necessitate new preclinical and clinical trials of a product. Such changes may also have
licensing implications depending on the legislated regulations in the country of manufacture, or the
countries where the product will be marketed and used.
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Fig. A5.10
Schematic diagram for the regulatory evaluation and control of the quality, safety and
standards of production of antivenoms
Venom producer
specifications
AUDITS
Plasma producer
GMP INSPECTION
specifications
Fractionator of antivenoms
specifications
Antivenom
Post-marketing
surveillance
Antivenom marketing file evaluation and authorization
all stages leading to the finished product, from production of plasma (including
venom sourcing and preparation, production animal sourcing, selection,
immunization and animal health control) to the collection and fractionation
of the plasma into the finished products and their control. Manufacturers of
antivenoms must maintain complete Site Master Files containing specific, factual
details of the application of GMP to production and quality control activities that
are undertaken at every site of operations linked to the products they produce.
Manufacturers should also maintain quality manuals that define and describe
the quality system, the scope and operations of the quality system at all levels
of production, management responsibilities, key quality systems processes
and safeguards. For individual products a product dossier in CTD format as
recommended by WHO and ICH may also be required. NRAs should make
full use of these three forms of production documentation in preparing for and
conducting site inspections and audits.
For local producers, the NRA should enforce the implementation of
GMP with the aim of ensuring the compliance of the manufacturer with the
existing provisions. It is the responsibility of the NRA inspector to ensure that
manufacturers adhere to the approved standards of GMP and quality assurance.
The inspection and enforcement of control measures for venom producers,
immune plasma producers, fractionation facilities and final product producers
and distributors should be carried out by officials representing the competent
NRA. They should be familiar with biological product technologies and trained
in GMP inspections.
Inspections should follow common inspection procedures. These include:
■■ an opening meeting with management and key personnel;
■■ a tour of the facility, including inspection of the main areas and
activities, such as:
(a) serpentariums;
(b) animal husbandry practices;
(c) animal identification and suitability for blood or plasma
collection;
(d) process of collection and storage of blood or plasma;
(e) plasma fractionation process;
(f ) testing and availability of test results for venoms, antivenoms
and raw materials;
(g) storage, transportation and shipment; and
(h) quality assurance (including internal audits and change
control);
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References
1. WHO Model List of Essential Medicines (15th list). Geneva: World Health Organization; 2007
(http://www.who.int/medicines/publications/08_ENGLISH_indexFINAL_EML15.pdf, accessed 13
February 2017).
2. Sells PG. Animal experimentation in snake venom research and in vitro alternatives. Toxicon.
2003;42(2):115–33.
3. Theakston RD, Warrell DA, Griffiths E. Report of a WHO workshop on the standardization and
control of antivenoms. Toxicon. 2003;41(5):541–57.
4. Gutiérrez JM, Herrera C. The analgesics morphine and tramadol do not alter the acute toxicity
induced by Bothrops asper snake venom in mice. Toxicon. 2014;81:54–7.
5. Von Behring E, Kitasato S. Über das Zustande-kommen der Diphtherie-Immunität und der
Tetanus-Immunität bei Thieren. Deutsche Medizinische Wochenschrif. 1890;16:1113–45.
6. Phisalix C, Bertrand G. Sur la propriété antitoxique du sang des animaux vaccinés contre le venin
de vipère. Comptes Rendus de la Société de Biologie. 1894;46:111–3.
7. Calmette A. Contribution à l’étude du venin des serpents. Immunisation des animaux et
traitement de l’envenimation. Annales de l’Institut Pasteur, VIII, 1894:275–91.
8. Calmette, A. Sur le venin des serpents et sur l’emploi du sérum antivenimeux dans la
thérapeutique des morsures venimeuses chez l’homme et chez les animaux. Annales de l’Institut
Pasteur. 1897;XII:214–37.
9. Pope CG. The action of proteolytic enzymes on the antitoxins and proteins in immune sera. I.
True digestion of the proteins. Br J Exp Pathol. 1939;20:132–49.
10. Pope CG. The action of proteolytic enzymes on the antitoxins and proteins in immmune sera. II.
Heat denaturation after partial enzyme action. Br J Exp Pathol. 1939;20:201–12.
11. Progress in the characterization of venoms and standardization of antivenoms. Geneva: World
Health Organization; 1981.
12. Raw I, Guidolin R, Higashi HG, Kelen EMA. Antivenins in Brazil: Preparation. In: Tu AT, editor.
Handbook of natural toxins. Volume 5: Reptile venoms and toxins. New York: Marcel Dekker;
1991:557–81.
13. Grandgeorge M, Véron JL, Lutsch C, Makula MF, Riffard P, et al. Preparation of improved F(abʹ)2
antivenoms. An example: new polyvalent European viper antivenom (equine). In: Bon C, Goyffon
M, editors. Envenomings and their treatments. Lyons: Fondation Marcel Mérieux; 1996:161–72.
14. Atkinson JP. The fractional precipitation of the globulin and albumin of normal horse’s serum
and diphtheria antitoxic serum, and the antitoxic strength of the precipitates. J Exp Med.
1900;5(1):67–76.
15. Hiss PH, Atkinson JP. Serum-globulin and diphtheric antitoxin – a comparative study of the
amount of globulin in normal and antitoxic sera, and the relation of the globulins to the antitoxic
bodies. J Exp Med. 1900;5(1):47–66.
16. Atkinson JF. A preliminary note on the fractional precipitation of the globulin and albumin
of normal horse’s serum and diphtheric antitoxic serum, and the antitoxic strength of the
precipitates. J Exp Med. 1899;4(5–6):649–54.
17. Avery OT. The distribution of the immune bodies occurring in antipneumococcus serum. J Exp
Med. 1915;21(2):133–45.
18. Gibson RB. The concentration of antitoxin for therapeutic use. J Biol Chem. 1906;1:161–70.
343
WHO Expert Committee on Biological Standardization Sixty-seventh report
19. Porter RR. The hydrolysis of rabbit y-globulin and antibodies with crystalline papain. Biochem J.
1959;73:119–26.
20. Gutiérrez JM, Leon G, Lomonte B. Pharmacokinetic-pharmacodynamic relationships of
immunoglobulin therapy for envenomation. Clin Pharmacokinet. 2003;42(8):721–41.
21. Rabies and envenomings: a neglected public health issue. Report of a consultative meeting.
Geneva: World Health Organization; 2007 (http://www.who.int/bloodproducts/animal_sera/
Rabies.pdf, accessed 13 February 2017).
22. Swaroop S, Grab B. Snakebite mortality in the world. Bull World Health Organ. 1954;10(1):35–76.
23. Chippaux JP. Snake-bites: appraisal of the global situation. Bull World Health Organ.
1998;76(5):515–24.
24. Snow RW, Bronzan R, Roques T, Nyamawi C, Murphy S, Marsh K. The prevalence and morbidity of
snake bite and treatment-seeking behaviour among a rural Kenyan population. Ann Trop Med
Parasitol. 1994;88(6):665–71.
25. Fox S, Rathuwithana AC, Kasturiratne A, Lalloo DG, de Silva HJ. Underestimation of snakebite
mortality by hospital statistics in the Monaragala District of Sri Lanka. Trans R Soc Trop Med Hyg.
2006;100(7):693–5.
26. Hati AK, Mandal M, De MK, Mukherjee H, Hati RN. Epidemiology of snake bite in the district of
Burdwan, West Bengal. J Indian Med Assoc. 1992;90(6):145–7.
27. Pugh RN, Theakston RD. Incidence and mortality on snake bite in savanna Nigeria. Lancet.
1980;2(8205):1181–3.
28. Sharma SK, Chappuis F, Jha N, Bovier PA, Loutan L, Koirala S. Impact of snake bites and determinants
of fatal outcomes in southeastern Nepal. Am J Trop Med Hyg. 2004;71(2):234–8.
29. Trape JF, Pison G, Guyavarch E, Mane Y. High mortality from snakebite in south-eastern Senegal.
Trans R Soc Trop Med Hyg. 2001;95(4):420–3.
30. Mohapatra B, Warrell DA, Suraweera W, Bhatia P, Dhingra N, Jotkar RM et al. Snakebite mortality in
India: a nationally representative mortality survey. PLoS Negl Trop Dis. 2011;5(4):e1018.
31. Rahman R, Abul Faiz M, Selim S, Rahman B, Basher A, Jones A et al. Annual incidence of snake
bite in rural Bangladesh. PLoS Negl Trop Dis. 2010;4(10):e860.
32. Ediriweera DS, Kasturiratne A, Pathmeswaran A, Gunawardena NK, Wijayawickrama BA,
Jayamanne SF et al. Mapping the risk of snakebite in Sri Lanka – a national survey with geospatial
WHO Technical Report Series, No. 1004, 2017
38. Ariaratnam CA, Thuraisingam V, Kularatne SAM, Sheriff MHR, Theakston RDG, Silva A et al. Frequent
and potentially fatal envenoming by hump-nosed pit vipers (Hypnale hypnale and H. nepa) in Sri
Lanka: lack of effective antivenom. Trans R Soc Trop Med Hyg. 2008;102(11):1120–6.
39. Chippaux JP, Williams V, White J. Snake venom variability: methods of study, results and
interpretation. Toxicon. 1991;29(11):1279–303.
40. Warrell DA. Geographical and intraspecies variation in the clinical manifestations of envenoming
by snakes. In: Thorpe RS, Wüster W, Malhotra A, editors. Venomous snakes: ecology, evolution and
snakebite. Oxford: Clarendon Press; 1997:189–203.
41. Fry BG, Winkel KD, Wickramaratna JC, Hodgson WC, Wüster W. Effectiveness of snake antivenom:
species and regional variation and its clinical impact. J Toxicol Toxin Reviews. 2003;22(1):23–34.
42. Raweerith R, Ratanabanangkoon K. Immunochemical and biochemical comparisons of equine
monovalent and polyvalent snake antivenoms. Toxicon. 2005;45(3):369–75.
43. Saravia P, Rojas E, Arce V, Guevara C, López JC, Chaves E et al. Geographic and ontogenic variability
in the venom of the neotropical rattlesnake Crotalus durissus: pathophysiological and therapeutic
implications. Rev Biol Trop. 2002;50(1):337–46.
44. Faure G, Bon C. Several isoforms of crotoxin are present in individual venoms from the South
American rattlesnake Crotalus durissus terrificus. Toxicon. 1987;25(2):229–34.
45. Creer S, Malhotra A, Thorpe RS, Stöcklin RS, Favreau PS, Chou W-H. Genetic and ecological
correlates of intraspecific variation in pitviper venom composition detected using matrix-assisted
laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and isoelectric focusing.
J Mol Evol. 2003;56(3):317–29.
46. Tan KY, Tan CH, Sim SM, Fung SY, Tan NH. Geographical venom variations of the Southeast Asian
monocled cobra (Naja kaouthia): venom-induced neuromuscular depression and antivenom
neutralization. Comp Biochem Physiol C Toxicol Pharmacol. 2016;185–186:77–86.
47. Tan KY, Tan CH, Fung SY, Tan NH. Venomics, lethality and neutralization of Naja kaouthia (monocled
cobra) venoms from three different geographical regions of Southeast Asia. J Proteomics.
2015;120:105–25.
48. Calvete JJ, Sanz L, Pérez A, Borges A, Vargas AM, Lomonte B et al. Snake population venomics
and antivenomics of Bothrops atrox: Paedomorphism along its transamazonian dispersal
and implications of geographic venom variability on snakebite management. J Proteomics.
2011;74(4):510–27.
49. Reichenbach-Klinke H-H, Elkan E. Diseases of reptiles. Neptune (NJ): TFH Publications; 1965.
50. Frye FL. Reptile care. An atlas of diseases and treatments. Two volumes. Neptune (NJ): TFH
Publications; 1991.
51. Cooper JE, Jackson OF. Diseases of the reptilia. Two volumes. London: Academic Press; 1981.
52. Shortridge KF, Ng MH, Oya A, Kobayashi M, Munro R, Wong F et al. Arbovirus infections in reptiles:
immunological evidence for a high incidence of Japanese encephalitis virus in the cobra Naja
naja. Trans R Soc Trop Med Hyg. 1974;68(6):454–60.
53. Leloup P. Various aspects of venomous snake breeding on a large scale. Acta Zool Pathol Antverp.
1984;78:177–98.
54. Mitchell MA. Snake care and husbandry. Vet Clin Exot Anim. 2004;7:421–46.
55. Chanhome L, Jintakune P, Wilde H, Cox MJ. Venomous snake husbandry in Thailand. Wilderness
Environ Med. 2001;12(1):17–23.
56. Gutiérrez JM, Chaves F, Bolaños R. Comparative study of venoms of newborn and adult specimens
of Bothrops asper. Rev Biol Trop. 1981;28(2):341–51.
345
WHO Expert Committee on Biological Standardization Sixty-seventh report
57. Furtado MFD, Maruyama M, Kamiguti AS, Antonio LC. Comparative study of nine Bothrops snake
venoms from adult female snakes and their offspring. Toxicon. 1991;29(2):219–26.
58. Alape-Girón A, Sanz L, Escolano J, Flores-Díaz M, Madrigal M, Sasa M et al. Snake venomics of
the lancehead pitviper Bothrops asper: geographic, individual, and ontogenetic variations.
J Proteome Res. 2008;7(8):3556–71.
59. Chacón D, Rodríguez S, Arias J, Solano G, Bonilla F, Gómez A. Maintaining coral snakes (Micrurus
nigrocinctus, Serpentes: Elapidae) for venom production on an alternative fish-based diet. Toxicon.
2012;60(3):249–53.
60. Goebel-Stengel M, Stengel A, Taché Y, Reeve Jr JR. The importance of using the optimal
plasticware and glassware in studies involving peptides. Anal Biochem. 2011;414(1):38–46.
61. Kurnik D, Haviv Y, Kochva E. A snake bite by the Burrowing Asp, Atractaspis engaddensis. Toxicon.
1999;37(1):223–7.
62. Harvey DS, Lentini AM, Cedar K, Weatherhead PJ. Moving massasaugas: insight into rattlesnake
relocation using Sistrurus c. catenatus. Herpetol Conserv Bio. 2014;9(1):67–75.
63. Roe JH, Frank MR, Gibson SE, Attum O, Kingsbury BA. No place like home: an experimental
comparison of reintroduction strategies using snakes. J Appl Ecol. 2010;47(6):1253–61.
64. Butler H, Malone B, Clemann N. The effects of translocation on the spatial ecology of tiger snakes
(Notechis scutatus) in a suburban landscape. Wildlife Res. 2005;32(2):165–71.
65. Reinert HK, Rupert RR. Impacts of translocation on behavior and survival of timber rattlesnakes,
Crotalus horridus. J Herpetol. 1999;33(1):45–61.
66. Powell RL, Sanchez EE, Pérez JC. Farming of venom: survey of snake venom extraction facilities
worldwide. Appl Herpetol. 2006;3(1):1–10.
67. Nishioka SA, Silveira PVP, Peixoto-Filho FM, Jorge MT, Sandoz A. Occupational injuries with
captive lance-headed vipers (Bothrops moojeni): experience from a snake farm in Brazil. Trop Med
Int Health. 2000;5(7):507–10.
68. de Medeiros CR, Barbaro KC, Lira MS, França FOS, Zaher VL, Kokron CM et al. Predictors of Bothrops
jararaca venom allergy in snake handlers and snake venom handlers. Toxicon. 2008;51(4):672–80.
69. Warrell DA. Envenoming, poisoning, hypersensitivity and injuries caused by animals. In: Warrell
DA, Cox TM, Firth JD, editors. Oxford textbook of medicine. Oxford: Oxford University Press; 2005.
70. Wüster W, McCarthy CJ. Venomous snake systematics: implications for snakebite treatment and
WHO Technical Report Series, No. 1004, 2017
toxinology. In: Bon C, Goyffon M, editors. Envenomings and their treatments. Lyons: Fondation
Marcel Mérieux; 1996:13–23.
71. León G, Segura A, Gomez A, Hernández A, Navarro D, Villalta M et al. Industrial production
and quality control of snake antivenoms. In: Gopalakrishnakone P, Calvete JJ, editors. Venom
genomics and proteomics. Dordrecht: Springer; 2016:1–22.
72. Angulo Y, Estrada R, Gutiérrez JM. Clinical and laboratory alterations in horses during
immunization with snake venoms for the production of polyvalent (Crotalinae) antivenom.
Toxicon. 1997;35(1):81–90.
73. Malikides N, Hodgson JL, Rose RJ, Hodgson DR. Cardiovascular, haematological and biochemical
responses after large volume blood collection in horses. Vet J. 2001;162(1):44–55.
74. Landon J, Woolley JA, McLean C. Antibody production in the hen. In: Landon J, Chard T, editors.
Therapeutic antibodies. London: Springer-Verlag; 1995:47–68.
75. Landon J, Smith D. Merits of sheep antisera for antivenom manufacture. J Toxicol Toxin Reviews.
2003;22:15–22.
346
Annex 5
76. Hanly WC, Artwohl JE, Bennett BT. Review of polyclonal antibody production procedures in
mammals and poultry. ILAR J. 1995;37(3):93–118.
77. WHO Guidelines on tissue infectivity distribution in transmissible spongiform encephalopathies.
Geneva: World Health Organization; 2006 (http://www.who.int/bloodproducts/cs/TSEPUBLISHED
REPORT.pdf, accessed 14 February 2017).
78. Moroz-Perlmutter C, Goldblum N, de Vries A, Gitter S. Detoxification of snake venoms and venom
fractions by formaldehyde. Exp Biol Med (Maywood). 1963;112(3):595–8.
79. Freitas TV, Fortes-Dias CL, Diniz CR, Velarde DT, Freitas CF. Immunization of horses with Crotalus
durissus terrificus (South American rattlesnake) venom. A comparison of four different procedures.
Braz J Med Biol Res. 1991;24(3):281–90.
80. Reed SG, Hsu F-C, Carter D, Orr MT. The science of vaccine adjuvants: advances in TLR4 ligand
adjuvants. Curr Opin Immunol. 2016;41:85–90.
81. Pratanaphon R, Akesowan S, Khow O, Sriprapat S, Ratanabanangkoon K. Production of highly
potent horse antivenom against the Thai cobra (Naja kaouthia). Vaccine. 1997;15(14):1523–8.
82. Chotwiwatthanakun C, Pratanaphon R, Akesowan S, Sriprapat S, Ratanabanangkoon K. Production
of potent polyvalent antivenom against three elapid venoms using a low dose, low volume,
multi-site immunization protocol. Toxicon. 2001;39(10):1487–94.
83. Quality assurance of pharmaceuticals: a compendium of guidelines and related materials.
Volume 2, Second updated edition. Good manufacturing practices and inspection. Geneva:
World Health Organization; 2007 (http://www.who.int/medicines/areas/quality_safety/quality_
assurance/QualityAssurancePharmVol2.pdf, accessed 14 February 2017).
84. Guidelines on viral inactivation and removal procedures intended to assure the viral safety of
human blood plasma products. In: WHO Expert Committee on Biological Standardization: fifty-
second report. Geneva: World Health Organization; 2004: Annex 4 (WHO Technical Report Series,
No. 924; http://www.who.int/bloodproducts/publications/WHO_TRS_924_A4.pdf, accessed 14
February 2017).
85. Recommendations for the production, control and regulation of human plasma for fractionation.
In: WHO Expert Committee on Biological Standardization: fifty-sixth report. Geneva: World
Health Organization; 2007: Annex 4 (WHO Technical Report Series, No. 941; http://www.who.int/
bloodproducts/publications/TRS941Annex4blood.pdf, accessed 14 February 2017).
86. Note for guidance on production and quality control of animal immunoglobulins and immunsera
for human use. London: European Agency for the Evaluation of Medicinal Products (EMEA),
CPMP; 2002:1–14.
87. Bolanos R, Cerdas L. Production and control of antivenin sera in Costa Rica. Bol Oficina Sanit
Panam. 1980;88(3):189–96.
88. Rojas G, Jimenez JM, Gutiérrez JM. Caprylic acid fractionation of hyperimmune horse plasma:
description of a simple procedure for antivenom production. Toxicon. 1994;32(3):351–63.
89. Otero R, Gutiérrez JM, Rojas G, Núñez V, Díaz A, Miranda E et al. A randomized blinded clinical
trial of two antivenoms, prepared by caprylic acid or ammonium sulphate fractionation of IgG,
in Bothrops and Porthidium snake bites in Colombia: correlation between safety and biochemical
characteristics of antivenoms. Toxicon. 1999;37(6):895–908.
90. Steinbuch M, Audran R. The isolation of IgG from mammalian sera with the aid of caprylic acid.
Arch Biochem Biophys. 1969;134(2):279–84.
91. Gutiérrez JM, Rojas E, Quesada L, León G, Núñez J, Laing GD et al. Pan-African polyspecific
antivenom produced by caprylic acid purification of horse IgG: an alternative to the antivenom
crisis in Africa. Trans R Soc Trop Med Hyg. 2005;99(6):468–75.
347
WHO Expert Committee on Biological Standardization Sixty-seventh report
92. dos Santos MC, D’Império Lima MR, Furtado GC, Colletto GM, Kipnis TL, Dias da Silva W. Purification
of F(abʹ)2 anti-snake venom by caprylic acid: a fast method for obtaining IgG fragments with high
neutralization activity, purity and yield. Toxicon. 1989;27(3):297–303.
93. Otero-Patiño R, Cardoso JL, Higashi HG, Nunez V, Diaz A, Toro MF et al. A randomized, blinded,
comparative trial of one pepsin-digested and two whole IgG antivenoms for Bothrops snake bites
in Uraba, Colombia. The Regional Group on Antivenom Therapy Research (REGATHER). Am J Trop
Med Hyg. 1998;58(2):183–9.
94. Abubakar IS, Abubakar SB, Habib AG, Nasidi A, Durfa N, Yusuf PO et al. Randomised controlled
double-blind non-inferiority trial of two antivenoms for saw-scaled or carpet viper (Echis
ocellatus) envenoming in Nigeria. PLoS Negl Trop Dis. 2010;4(7):e767.
95. Jones RG, Landon J. A protocol for “enhanced pepsin digestion”: a step by step method for
obtaining pure antibody fragments in high yield from serum. J Immunol Methods. 2003;275(1–
2):239–50.
96. Raweerith R, Ratanabanangkoon K. Fractionation of equine antivenom using caprylic acid
precipitation in combination with cationic ion-exchange chromatography. J Immunol Methods.
2003;282(1–2):63–72.
97. Al-Abdulla I, Garnvwa JM, Rawat S, Smith DS, Landon J, Nasidi A. Formulation of a liquid ovine
Fab-based antivenom for the treatment of envenomation by the Nigerian carpet viper (Echis
ocellatus). Toxicon. 2003;42(4):399–404.
98. Saetang T, Treamwattana N, Suttijitpaisal P, Ratanabanangkoon K. Quantitative comparison on
the refinement of horse antivenom by salt fractionation and ion-exchange chromatography. J
Chromatogr B Biomed Sci Appl. 1997;700(1–2):233–9.
99. Sullivan JB Jr, Russell FE. Isolation and purification of antibodies to rattlesnake venom by affinity
chromatography. Proc West Pharmacol Soc. 1982;25:185–92.
100. Magos L. Review on the toxicity of ethylmercury, including its presence as a preservative in
biological and pharmaceutical products. J Appl Toxicol. 2001;21(1):1–5.
101. Guidelines on regulatory expectations related to the elimination, reduction or replacement
of thiomersal in vaccines. In: WHO Expert Committee on Biological Standardization: fifty-third
report. Geneva: World Health Organization; 2004: Annex 4 (WHO Technical Report Series, No. 926;
http://www.who.int/biologicals/publications/trs/areas/vaccines/thiomersal/Annex%204%20(95-
102)TRS926thiomersal.pdf, accessed 14 February 2017).
102. Pikal MJ. Freeze drying. In: Encyclopedia of pharmaceutical technology. New York: Marcel Dekker;
WHO Technical Report Series, No. 1004, 2017
2002:1299–326.
103. Herrera M, Tattini V Jr, Pitombo RNM, Gutiérrez JM, Borgognoni C, Vega-Baudrit J et al. Freeze-dried
snake antivenoms formulated with sorbitol, sucrose or mannitol: comparison of their stability in
an accelerated test. Toxicon. 2014;90:56–63.
104. Meyer WP, Habib AG, Onayade AA, Yakubu A, Smith DC, Nasidi A et al. First clinical experiences
with a new ovine Fab Echis ocellatus snake bite antivenom in Nigeria: randomized comparative
trial with Institute Pasteur Serum (IPSER) Africa antivenom. Am J Trop Med Hyg. 1997;56(3):
291–300.
105. Ariaratnam CA, Meyer WP, Perera G, Eddleston M, Kuleratne SA, Attapattu W et al. A new
monospecific ovine Fab fragment antivenom for treatment of envenoming by the Sri Lankan
Russell’s viper (Daboia Russelii Russelii): a preliminary dose-finding and pharmacokinetic study.
Am J Trop Med Hyg. 1999;61(2):259–65.
106. Scherrmann JM. Antibody treatment of toxin poisoning – recent advances. J Toxicol Clin Toxicol.
1994;32(4):363–75.
348
Annex 5
125. Lundblad JL, Seng RL. Inactivation of lipid-enveloped viruses in proteins by caprylate. Vox Sang.
1991;60(2):75–81.
126. Burnouf T, Terpstra F, Habib G, Seddik S. Assessment of viral inactivation during pH 3.3 pepsin
digestion and caprylic acid treatment of antivenoms. Biologicals. 2007;35(4):329–34.
127. El-Ekiaby M, Vargas M, Sayed M, Gorgy G, Goubran H, Radosevic M et al. Minipool caprylic
acid fractionation of plasma using disposable equipment: a practical method to enhance
immunoglobulin supply in developing countries. PLoS Negl Trop Dis. 2015;9(2):e0003501.
128. Mpandi M, Schmutz P, Legrand E, Duc R, Geinoz J, Henzelin-Nkubana C et al. Partitioning and
inactivation of viruses by the caprylic acid precipitation followed by a terminal pasteurization
in the manufacturing process of horse immunoglobulins. Biologicals. 2007;35(4):335–41.
129. Lazar A, Epstein E, Lustig S, Barnea A, Silberstein L, Reuveny S. Inactivation of West-Nile virus
during peptic cleavage of horse plasma IgG. Biologicals. 2002;30(2):163–5.
130. Cameron-Smith R, Miloradovic L, Cheyne I, Healy K. The removal of viruses during the purification
of equine antisera using filtration aids Hyflo Super-Cel™ and Fulmont™ Super A. Biologicals.
2000;28(3):169–74.
131. Burnouf T, Radosevich M, Goubran HA, Willkommen H. Place of nanofiltration for assuring viral
safety of biologicals. Curr Nanosci. 2005;1(3):189–201.
132. Caricati CP, Oliveira-Nascimento L, Yoshida JT, Stephano MA, Caricati ATP. Safety of snake
antivenom immunoglobulins: efficacy of viral inactivation in a complete downstream process.
Biotechnol Prog. 2013;29(4):972–9.
133. Tuck S, Ng R. Protein assays. In: Rodriguez-Diaz R, Wehr T, editors. Analytical techniques for
biopharmaceutical development. New York: Marcel Dekker; 2005:14–24.
134. Segura A, Herrera M, Villalta M, Vargas M, Gutiérrez JM, León G. Assessment of snake antivenom
purity by comparing physicochemical and immunochemical methods. Biologicals. 2013;41(2):
93–7.
135. Solano G, Gomez A, Leon G. Assessing endotoxins in equine-derived snake antivenoms:
comparison of the USP pyrogen test and the Limulus amoebocyte lysate assay (LAL). Toxicon.
2015;105:13–8.
136. Sanchez LV, Pla D, Herrera M, Chippaux JP, Calvete JJ, Gutiérrez JM. Evaluation of the preclinical
efficacy of four antivenoms, distributed in sub-Saharan Africa, to neutralize the venom of the
carpet viper, Echis ocellatus, from Mali, Cameroon, and Nigeria. Toxicon. 2015;106:97–107.
137. Pla D, Gutiérrez JM, Calvete JJ. Second generation snake antivenomics: comparing immunoaffinity
WHO Technical Report Series, No. 1004, 2017
143. Rojas E, Quesada L, Arce V, Lomonte B, Rojas G, Gutiérrez JM. Neutralization of four Peruvian
Bothrops sp. snake venoms by polyvalent antivenoms produced in Perú and Costa Rica: preclinical
assessment. Acta Trop. 2005;93(1):85–95.
144. Reid HA, Theakston RD. The management of snake bite. Bull World Health Organ. 1983;61(6):
885–95.
145. Theakston RD, Reid HA. Development of simple standard assay procedures for the characterization
of snake venom. Bull World Health Organ. 1983;61(6):949–56.
146. Gutiérrez JM, Gené JA, Rojas G, Cerdas L. Neutralization of proteolytic and hemorrhagic activities
of Costa Rican snake venoms by a polyvalent antivenom. Toxicon. 1985;23(6):887–93.
147. Reid HA. Cobra-bites. Br Med J. 1964;2(5408):540–5.
148. Gutiérrez JM, León G, Rojas G, Lomonte B, Rucavado A, Chaves F. Neutralization of local tissue
damage induced by Bothrops asper (terciopelo) snake venom. Toxicon. 1998;36(11):1529–38.
149. Iddon D, Theakston RD, Ownby CL. A study of the pathogenesis of local skin necrosis induced
by Naja nigricollis (spitting cobra) venom using simple histological staining techniques. Toxicon.
1987;25(6):665–72.
150. Rivel M, Solano D, Herrera M, Vargas M, Villalta M, Segura A et al. Pathogenesis of dermonecrosis
induced by venom of the spitting cobra, Naja nigricollis: an experimental study in mice. Toxicon.
2016;119:171–9.
151. Laing GD, Theakston RDG, Leite RP, Dias da Silva WD, Warrell DA, BIASG (Butantan Institute
Antivenom Study Group). Comparison of the potency of three Brazilian Bothrops antivenoms
using in vivo rodent and in vitro assays. Toxicon. 1992;30(10):1219–25.
152. Theakston RDG. Characterization of venoms and standardization of antivenoms. In: Harris JB,
editor. Natural toxins, animal, plant and microbial. Oxford: Clarendon Press, Oxford Science
Publications; 1986:287–303.
153. Gene JA, Roy A, Rojas G, Gutiérrez JM, Cerdas L. Comparative study on coagulant, defibrinating,
fibrinolytic and fibrinogenolytic activities of Costa Rican crotaline snake venoms and their
neutralization by a polyvalent antivenom. Toxicon. 1989;27(8):841–8.
154. Gutiérrez JM, Romero M, Núñez J, Chaves F, Borkow G, Ovadia M. Skeletal muscle necrosis and
regeneration after injection of BaH1, a hemorrhagic metalloproteinase isolated from the venom
of the snake Bothrops asper (terciopelo). Exp Mol Pathol. 1995;62(1):28–41.
155. Gutiérrez JM, Rojas G, Jorge da Silva N, Núñez J. Experimental myonecrosis induced by the
venoms of South American Micrurus (coral snakes). Toxicon. 1992;30(10):1299–302.
156. Alam JM, Ali SA, Faridi N, Alam SM. Myotoxic characteristics of snake venoms: myonecrosis
induced in albino rats by crude venoms and the purified myotoxic component, phospholipase A2.
J Coll Physicians Surg Pak. 2002;12(7):398–402.
157. Ginsborg BL, Warriner J. The isolated chick biventer cervicis nerve-muscle preparation. Br J
Pharmacol Chemother. 1960;15:410–11.
158. Harvey AL, Barfaraz A, Thomson E, Faiz A, Preston S, Harris JB. Screening of snake venoms for
neurotoxic and myotoxic effects using simple in vitro preparations from rodents and chicks.
Toxicon. 1994;32(3):257–65.
159. Bulbring E. Observations on the isolated phrenic nerve diaphragm preparation of the rat. Br J
Pharmacol Chemother. 1946;1:38–61.
160. Jones RG, Lee L, Landon J. The effects of specific antibody fragments on the “irreversible”
neurotoxicity induced by Brown snake (Pseudonaja) venom. Br J Pharmacol. 1999;126(3):581–4.
161. Crachi MT, Hammer LW, Hodgson WC. A pharmacological examination of venom from the
Papuan taipan (Oxyuranus scutellatus canni). Toxicon. 1999;37(12):1721–34.
351
WHO Expert Committee on Biological Standardization Sixty-seventh report
162. Kitchen I. Neuromuscular blocking drugs and the rat phrenic nerve hemidiaphragm preparation.
In: Textbook of in vitro pharmacology. Oxford: Blackwell Scientific; 2009:79–83.
163. Theakston RDG, Laing GD, Fielding CM, Freite Lascano A, Touzet J-M, Vallejo F et al. Treatment of
snake bites by Bothrops species and Lachesis muta in Ecuador: laboratory screening of candidate
antivenoms. Trans R Soc Trop Med Hyg. 1995;89(5):550–4.
164. Keegan HL, Weaver RE, Matsui T. Southeast Asian snakebite antivenin studies. Japan: 406th
Medical Laboratory, Control Department of Entomology, US Army Medical Command; 1964.
165. Warrell DA, Warrell MJ, Edgar W, Prentice CR, Mathison J, Mathison J. Comparison of Pasteur
and Behringwerke antivenoms in envenoming by the carpet viper (Echis carinatus). Br Med J.
1980;280(6214):607–9.
166. Rosenberger WF, Haines LM. Competing designs for phase I clinical trials: a review. Stat Med.
2002;21(18):2757–70.
167. Chippaux J-P, Stock RP, Massougbodji A. Antivenom safety and tolerance for the strategy of
snake envenomation management. In: Gopalakrishnakone P, Inagaki H, Mukherjee AK, Rahmy TR,
Vogel C-W, editors. Snake venoms. Dordrecht: Springer Science+Business Media; 2015:1–16.
168. Machin D, Campbell MJ, Tan SB, Tan SH. Sample size tables for clinical studies, third edition.
Chichester: Wiley Blackwell; 2009.
169. Visser LE, Kyei-Faried S, Belcher DW, Geelhoed DW, Schagen van Leeuwen J, van Roosmalen J.
Failure of a new antivenom to treat Echis ocellatus snake bite in rural Ghana: the importance of
quality surveillance. Trans R Soc Trop Med Hyg. 2008;102(5):445–50.
170. Armitage P. Sequential medical trials, second edition. New York: John Wiley; 1975.
171. Vakil BJ, Armitage P, Clifford RE, Laurence DR. Therapeutic trial of intracisternal human tetanus
immunoglobulin in clinical tetanus. Trans R Soc Trop Med Hyg. 1979;73(5):579–83.
172. Smalligan R, Cole J, Brito N, Laing GD, Mertz BL, Manock S et al. Crotaline snake bite in the
Ecuadorian Amazon: randomised double blind comparative trial of three South American
polyspecific antivenoms. BMJ. 2004;329:1129.
173. Ariaratnam CA, Sjöström L, Raziek Z, Abeyasinghe S, Kularatne M, Kodikara Arachchi RWK et al.
An open, randomized comparative trial of two antivenoms for the treatment of envenoming by
Sri Lankan Russell’s viper (Daboia russelii russelii). Trans R Soc Trop Med Hyg. 2001;95(1):74–80.
174. Warrell DA, Looareesuwan S, Theakston RD, Phillips RE, Chanthavanich P, Viravan C et al.
Randomized comparative trial of three monospecific antivenoms for bites by the Malayan pit
WHO Technical Report Series, No. 1004, 2017
179. Bush SP, Green SM, Moynihan JA, Hayes WK, Cardwell MD. Crotalidae polyvalent immune Fab
(ovine) antivenom is efficacious for envenomations by Southern Pacific rattlesnakes (Crotalus
helleri). Ann Emerg Med. 2002;40(6):619–24.
180. Warrell DA. Unscrupulous marketing of snake bite antivenoms in Africa and Papua New Guinea:
choosing the right product – “what’s in a name?”. Trans R Soc Trop Med Hyg. 2008;102(5):397–9.
181. Guidelines for national authorities on quality assurance for biological products. In: WHO
Expert Committee on Biological Standardization: forty-second report. Geneva: World Health
Organization; 1992: Annex 2 (WHO Technical Report Series, No. 822; http://apps.who.int/iris/
bitstream/10665/39431/1/WHO_TRS_822.pdf, accessed 14 February 2017).
182. Regulation and licensing of biological products in countries with newly developing regulatory
authorities. In: WHO Expert Committee on Biological Standardization: forty-fifth report. Geneva:
World Health Organization; 1995: Annex 1 (WHO Technical Report Series, No. 858; http://apps.
who.int/iris/bitstream/10665/36999/1/WHO_TRS_858.pdf, accessed 14 February 2017).
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Appendix 1
Worldwide distribution of medically important
venomous snakes
Venomous snakes are widely distributed, especially in tropical countries, from
sea level to altitudes of up to 4900 metres (Gloydius himalayanus). The European
adder (Vipera berus) enters the Arctic Circle, and the Argentine Yararanata
(Bothrops ammodytoides) occurs to 47 °S and is the most southerly occurring
venomous snake. No other venomous species occur in cold regions such as
the Arctic, Antarctic and north of around latitude 51 °N in North America
(Newfoundland, Nova Scotia).
This Appendix lists venomous snake species considered to represent the
greatest threat to public health in various countries, territories and other areas or
regions around the world. Only species which fall into one of the two categories
listed below are shown, and category listings are in alphabetical order according
to taxonomic family, genus and species. The intention in categorizing these
medically important snakes into two groups is to provide users of these WHO
Guidelines with a prioritized listing. Snakes in both Category 1 and Category 2
are species for which antivenom production is important; however species
listed in Category 1 within a country, territory or area should be considered as
being of highest priority for antivenom production on the basis that available
knowledge implicates them as being responsible for the greater burden in that
particular setting.
Definitions of the categories used in this listing are:
■■ CATEGORY 1 (CAT 1): Highest medical importance
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Cyprus:
Cat 1: None
Cat 2: Viperidae: Macrovipera lebetina
Egypt:
Cat 1: Elapidae: Naja haje; Viperidae: Cerastes cerastes; Echis coloratus (east),
Echis pyramidum
Cat 2: Atractaspididae: Atractaspis engaddensis (Sinai); Elapidae: Naja nubiae;
Walterinnesia aegyptia (Sinai); Viperidae: Pseudocerastes fieldi
Iraq:
Cat 1: Viperidae: Echis carinatus; Macrovipera lebetina
Cat 2: Elapidae: Walterinnesia morgani; Viperidae: Cerastes gasperettii;
Pseudocerastes fieldi, Pseudocerastes persicus
Israel:
Cat 1: Viperidae: Daboia palaestinae; Echis coloratus
Cat 2: Atractaspididae: Atractaspis engaddensis; Elapidae: Walterinnesia aegyptia;
Viperidae: Cerastes cerastes, Cerastes gasperettii; Pseudocerastes fieldi
Jordan:
Cat 1: Viperidae: Daboia palaestinae; Echis coloratus
Cat 2: Atractaspididae: Atractaspis engaddensis; Elapidae: Walterinnesia aegyptia;
Viperidae: Cerastes gasperettii; Macrovipera lebetina; Pseudocerastes fieldi
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Lebanon:
Cat 1: Viperidae: Daboia palaestinae; Macrovipera lebetina
Cat 2: None
Libya:
Cat 1: Elapidae: Naja haje; Viperidae: Cerastes cerastes; Echis pyramidum
Cat 2: Viperidae: Daboia deserti
Morocco:
Cat 1: Elapidae: Naja haje; Viperidae: Bitis arietans; Cerastes cerastes; Daboia
mauritanica
Cat 2: Viperidae: Echis leucogaster; Vipera latastei
Oman:
Cat 1: Atractaspididae: Atractaspis andersonii (south-west); Viperidae: Bitis arietans
(south-west); Echis coloratus (south-west), Echis carinatus, Echis omanensis
(north)
Cat 2: Elapidae: Naja arabica (south-west); Viperidae: Cerastes gasperettii;
Echis khosatzkii (south-west); Pseudocerastes persicus
Saudi Arabia:
Cat 1: Atractaspididae: Atractaspis andersonii (south-west);
Viperidae: Cerastes gasperettii; Echis coloratus, Echis borkini (south-west)
Cat 2: Atractaspididae: Atractaspis engaddensis (north-west); Elapidae: Naja arabica
(south-west); Walterinnesia aegyptia (west), Walterinnesia morgani (central
and south); Viperidae: Bitis arietans (south-west); Cerastes cerastes (south-
west); Pseudocerastes fieldi
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Tunisia:
Cat 1: Viperidae: Daboia mauritanica
Cat 2: Elapidae: Naja haje; Viperidae: Cerastes cerastes; Daboia deserti; Echis
leucogaster; Macrovipera lebetina; Vipera latastei
Turkey:
Cat 1: Viperidae: Macrovipera lebetina; Montivipera xanthina
Cat 2: Elapidae: Walterinnesia morgani (south); Viperidae: Montivipera raddei;
Vipera ammodytes, Vipera eriwanensis, Vipera spp.
Western Sahara:
Cat 1: Viperidae: Cerastes cerastes
Cat 2: Elapidae: Naja haje; Viperidae: Bitis arietans
Yemen:
Cat 1: Atractaspididae: Atractaspis andersonii; Elapidae: Naja arabica;
Viperidae: Bitis arietans; Echis borkini, Echis coloratus
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Burundi:
Cat 1: Elapidae: Naja nigricollis, Naja melanoleuca; Viperidae: Bitis arietans
Cat 2: Atractaspididae: Atractaspis bibronii, Atractaspis irregularis;
Colubridae: Dispholidus typus; Thelotornis mossambicanus;
Elapidae: Dendroaspis jamesoni; Viperidae: Bitis gabonica, Bitis nasicornis
Chad:
Cat 1: Elapidae: Naja haje, Naja nigricollis; Viperidae: Bitis arietans (south);
Echis ocellatus (south)
Cat 2: Colubridae: Dispholidus typus; Elapidae: Naja katiensis, Naja nubiae;
Viperidae: Cerastes cerastes
The medical importance of this species may be higher in the primary forest zone of the south-western
1
Central African Republic, and in some secondary forest mosaic zones elsewhere in the Central African
Republic.
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Equatorial Guinea:
Cat 1: Elapidae: Dendroaspis jamesoni; Naja melanoleuca; Viperidae: Bitis gabonica,
Bitis nasicornis
Cat 2: Atractaspididae: Atractaspis irregularis; Colubridae: Thelotornis kirtlandii;
Elapidae: Naja annulata; Pseudohaje goldii; Viperidae: Atheris squamigera
Gabon:
Cat 1: Elapidae: Dendroaspis jamesoni; Naja melanoleuca, Naja nigricollis;
Viperidae: Bitis gabonica, Bitis nasicornis
Cat 2: Atractaspididae: Atractaspis irregularis; Colubridae: Thelotornis kirtlandii;
Elapidae: Naja annulata; Pseudohaje goldii; Viperidae: Atheris squamigera;
Bitis arietans
Rwanda:
Cat 1: Elapidae: Dendroaspis jamesoni; Naja nigricollis; Viperidae: Bitis arietans
Cat 2: Atractaspididae: Atractaspis bibronii, Atractaspis irregularis;
Colubridae: Dispholidus typus; Thelotornis kirtlandii;
Elapidae: Dendroaspis polylepis; Naja annulata, Naja melanoleuca;
Pseudohaje goldii; Viperidae: Bitis gabonica, Bitis nasicornis
Eritrea:
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Ethiopia:
Cat 1: Elapidae: Dendroaspis polylepis; Naja ashei (south-east), Naja haje,
Naja nigricollis; Viperidae: Bitis arietans; Echis pyramidum
Cat 2: Atractaspididae: Atractaspis fallax, Atractaspis irregularis (Mt Bizen);
Colubridae: Dispholidus typus; Elapidae: Naja melanoleuca, Naja pallida;
Viperidae: Bitis parviocula, Bitis harenna
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Kenya:
Cat 1: Elapidae: Dendroaspis angusticeps, Dendroaspis polylepis; Naja ashei (north
& east), Naja haje, Naja nigricollis; Viperidae: Bitis arietans; Echis pyramidum
Cat 2: Atractaspididae: Atractaspis bibronii, Atractaspis fallax, Atractaspis irregularis;
Colubridae: Dispholidus typus; Thelotornis mossambicanus,
Thelotornis usambaricus (east coast); Elapidae: Dendroaspis jamesoni;
Naja melanoleuca (west and coastal forest), Naja pallida (north and east);
Pseudohaje goldii; Viperidae: Atheris squamigera; Bitis nasicornis, Bitis gabonica
(west)
Malawi:
Cat 1: Elapidae: Dendroaspis angusticeps, Dendroaspis polylepis; Naja annulifera,
Naja mossambica, Naja nigricollis; Viperidae: Bitis arietans
Cat 2: Atractaspididae: Atractaspis bibronii; Colubridae: Dispholidus typus;
Thelotornis capensis, Thelotornis mossambicanus; Elapidae: Naja melanoleuca;
Viperidae: Proatheris superciliaris
Mozambique:
Cat 1: Elapidae: Dendroaspis angusticeps, Dendroaspis polylepis; Naja annulifera,
Naja mossambica; Viperidae: Bitis arietans, Bitis gabonica
Cat 2: Atractaspididae: Atractaspis bibronii; Colubridae: Dispholidus typus;
Thelotornis capensis, Thelotornis mossambicanus;
Elapidae: Hemachatus haemachatus; Naja melanoleuca;
Viperidae: Proatheris superciliaris
Somalia:
Cat 1: Elapidae: Dendroaspis polylepis; Naja ashei (south), Naja haje;
Viperidae: Bitis arietans; Echis pyramidum
Cat 2: Atractaspididae: Atractaspis fallax; Colubridae: Dispholidus typus;
Thelotornis mossambicanus; Elapidae: Naja pallida, Naja melanoleuca;
Viperidae: Echis hughesi (north)
South Sudan:
Cat 1: Elapidae: Naja haje, Naja nigricollis; Viperidae: Bitis arietans;
Echis pyramidum
Cat 2: Atractaspididae: Atractaspis fallax, Atractaspis irregularis;
Colubridae: Dispholidus typus; Elapidae: Dendroaspis jamesoni,
Dendroaspis polylepis; Naja melanoleuca, Naja nubiae, Naja pallida;
Viperidae: Bitis gabonica, Bitis nasicornis
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Sudan:
Cat 1: Elapidae: Naja haje; Viperidae: Bitis arietans; Echis pyramidum
Cat 2: Colubridae: Dispholidus typus; Elapidae: Dendroaspis polylepis (east);
Naja nubiae; Viperidae: Cerastes cerastes, Echis coloratus (east)
Uganda:
Cat 1: Elapidae: Naja ashei (north-east), Naja haje (north), Naja nigricollis;
Dendroaspis jamesoni, Dendroaspis polylepis; Viperidae: Bitis arietans,
Bitis gabonica
Cat 2: Atractaspididae: Atractaspis irregularis; Colubridae: Dispholidus typus;
Thelotornis kirtlandii; Elapidae: Naja melanoleuca; Pseudohaje goldii;
Viperidae: Atheris squamigera; Bitis nasicornis
Zambia:
Cat 1: Elapidae: Dendroaspis polylepis; Naja anchietae, Naja annulifera,
Naja mossambica, Naja nigricollis; Viperidae: Bitis arietans, Bitis gabonica
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Lesotho:
Cat 1: Elapidae: Naja nivea; Viperidae: Bitis arietans
Cat 2: Elapidae: Hemachatus haemachatus
Namibia:
Cat 1: Elapidae: Dendroaspis polylepis; Naja anchietae, Naja nivea (central and
southern), Naja mossambica (north-east), Naja nigricincta;
Viperidae: Bitis arietans
Cat 2: Atractaspididae: Atractaspis bibronii; Colubridae: Dispholidus typus;
Thelotornis capensis; Elapidae: Naja nigricollis (Caprivi)
South Africa:
Cat 1: Elapidae: Dendroaspis angusticeps (Natal), Dendroaspis polylepis;
Naja annulifera (north-east), Naja nivea, Naja mossambica (north-east);
Viperidae: Bitis arietans
Cat 2: Atractaspididae: Atractaspis bibronii; Colubridae: Dispholidus typus;
Thelotornis capensis; Elapidae: Hemachatus haemachatus; Naja melanoleuca
(KwaZulu-Natal), Naja nigricincta (north-west); Viperidae: Bitis gabonica
(KwaZulu-Natal)
Swaziland:
Cat 1: Elapidae: Dendroaspis polylepis; Naja annulifera, Naja mossambica;
Viperidae: Bitis arietans
Cat 2: Atractaspididae: Atractaspis bibronii; Colubridae: Dispholidus typus;
Thelotornis capensis; Elapidae: Hemachatus haemachatus
Zimbabwe:
Cat 1: Elapidae: Dendroaspis polylepis; Naja anchietae (west), Naja annulifera,
Naja mossambica; Viperidae: Bitis arietans
Cat 2: Atractaspididae: Atractaspis bibronii; Colubridae: Dispholidus typus;
Thelotornis capensis, Thelotornis mossambicanus;
Elapidae: Dendroaspis angusticeps (east); Hemachatus haemachatus (Nyanga
Mountains); Naja melanoleuca (east); Viperidae: Bitis gabonica (east)
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Burkina Faso:
Cat 1: Elapidae: Naja nigricollis, Naja katiensis; Viperidae: Bitis arietans;
Echis ocellatus
Cat 2: Colubridae: Dispholidus typus; Elapidae: Dendroaspis polylepis;
Naja melanoleuca, Naja senegalensis; Viperidae: Echis leucogaster
Cameroon:
Cat 1: Elapidae: Dendroaspis jamesoni; Naja haje, Naja nigricollis, Naja
melanoleuca; 2 Viperidae: Bitis arietans, Bitis gabonica, Bitis nasicornis;
Echis ocellatus
Cat 2: Atractaspididae: Atractaspis irregularis; Colubridae: Dispholidus typus;
Thelotornis kirtlandii; Elapidae: Dendroaspis polylepis; Naja annulata,
Naja katiensis; Pseudohaje goldii; Viperidae: Atheris broadleyi (East Province),
Atheris squamigera
Côte d’Ivoire:
Cat 1: Elapidae: Dendroaspis viridis; Naja nigricollis, Naja melanoleuca,
Naja senegalensis; Viperidae: Bitis arietans, Bitis nasicornis, Bitis rhinoceros;
Echis ocellatus
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Gambia:
Cat 1: Elapidae: Dendroaspis viridis; Naja nigricollis; Viperidae: Bitis arietans;
Echis jogeri
Cat 2: Colubridae: Dispholidus typus; Elapidae: Naja katiensis, Naja melanoleuca,
Naja senegalensis
This large, highly venomous snake is common in forested areas of south-west Cameroon and a high
2
burden of injury may be expected, although clinical data with direct attribution are not yet available.
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Ghana:
Cat 1: Elapidae: Dendroaspis viridis; Naja nigricollis, Naja senegalensis;
Viperidae: Bitis arietans; Echis ocellatus
Cat 2: Atractaspididae: Atractaspis irregularis; Colubridae: Dispholidus typus,
Thelotornis kirtlandii; Elapidae: Naja katiensis, Naja melanoleuca; 3
Pseudohaje goldii, Pseudohaje nigra; Viperidae: Atheris chlorechis;
Bitis nasicornis, Bitis rhinoceros
Guinea:
Cat 1: Elapidae: Dendroaspis polylepis, Dendroaspis viridis; Naja katiensis,
Naja nigricollis, Naja melanoleuca, Naja senegalensis;
Viperidae: Bitis arietans; Echis jogeri
Cat 2: Atractaspididae: Atractaspis irregularis; Colubridae: Dispholidus typus;
Thelotornis kirtlandii; Elapidae: Pseudohaje nigra; Viperidae: Atheris chlorechis;
Bitis nasicornis, Bitis rhinoceros
Guinea-Bissau:
Cat 1: Elapidae: Dendroaspis viridis; Naja nigricollis, Naja melanoleuca,
Naja senegalensis; Viperidae: Bitis arietans; Echis jogeri
Cat 2: Colubridae: Dispholidus typus; Thelotornis kirtlandii; Viperidae: Bitis rhinoceros
Liberia:
Cat 1: Elapidae: Dendroaspis viridis; Naja melanoleuca, Naja nigricollis
Cat 2: Atractaspididae: Atractaspis irregularis; Colubridae: Thelotornis kirtlandii;
Elapidae: Pseudohaje nigra; Viperidae: Atheris chlorechis; Bitis nasicornis,
Bitis rhinoceros
Mali:
Cat 1: Elapidae: Naja katiensis, Naja nigricollis, Naja senegalensis;
Viperidae: Bitis arietans; Echis jogeri (west), Echis leucogaster, Echis ocellatus
Cat 2: Colubridae: Dispholidus typus; Elapidae: Naja melanoleuca;
Viperidae: Cerastes cerastes
Mauritania:
Cat 1: Elapidae: Naja senegalensis (south-east); Viperidae: Cerastes cerastes;
Echis leucogaster
Cat 2: Viperidae: Bitis arietans
The medical importance of this species may be higher in the forested zone of southern Ghana.
3
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Niger:
Cat 1: Elapidae: Naja nigricollis; Viperidae: Bitis arietans; Echis leucogaster,
Echis ocellatus
Cat 2: Colubridae: Dispholidus typus; Elapidae: Naja haje (south-central),
Naja katiensis, Naja nubiae; Naja senegelensis (south-west);
Viperidae: Cerastes cerastes
Nigeria:
Cat 1: Elapidae: Dendroaspis jamesoni; Naja haje (north-east), Naja nigricollis;
Viperidae: Bitis arietans, Bitis gabonica; Echis ocellatus
Cat 2: Atractaspididae: Atractaspis irregularis; Colubridae: Dispholidus typus;
Thelotornis kirtlandii; Elapidae: Naja katiensis, Naja melanoleuca,4
Naja senegalensis (north-west); Pseudohaje goldii, Pseudohaje nigra;
Viperidae: Atheris squamigera; Bitis nasicornis; Echis leucogaster (north)
Senegal:
Cat 1: Elapidae: Naja katiensis, Naja nigricollis; Viperidae: Bitis arietans;
Echis leucogaster, Echis jogeri
Cat 2: Colubridae: Dispholidus typus; Elapidae: Dendroaspis polylepis,
Dendroaspis viridis; Naja melanoleuca, Naja senegalensis
Sierra Leone:
Cat 1: Elapidae: Dendroaspis viridis; Naja nigricollis; Viperidae: Bitis arietans
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4
The medical importance of this species may be higher in the southern rainforest belt of Nigeria, from
Ibadan in the west to Oban and Eket in the east, and in the forested southern quarter of Sierra Leone.
5
The medical importance of this species may be higher in the forested southern quarter of Sierra Leone.
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Togo:
Cat 1: Elapidae: Naja nigricollis, Naja senegalensis; Viperidae: Bitis arietans (south);
Echis ocellatus
Cat 2: Atractaspididae: Atractaspis irregularis; Colubridae: Dispholidus typus;
Thelotornis kirtlandii; Elapidae: Dendroaspis jamesoni, Dendroaspis viridis;
Naja katiensis, Naja melanoleuca; Pseudohaje goldii, Pseudohaje nigra;
Viperidae: Atheris chlorechis; Bitis nasicornis, Bitis rhinoceros
Azerbaijan:
Cat 1: Viperidae: Macrovipera lebetina
Cat 2: Viperidae: Gloydius halys; Vipera eriwanensis, Vipera spp.
Georgia:
Cat 1: Viperidae: Macrovipera lebetina; Vipera ammodytes
Cat 2: Viperidae: Vipera kaznakovi, Vipera renardi, Vipera spp.
Mongolia:
Cat 1: Viperidae: Gloydius halys
Cat 2: Viperidae: Vipera berus, Vipera renardi
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East Asia
China:
China mainland
Cat 1: Elapidae: Bungarus multicinctus; Naja atra;
Viperidae: Trimeresurus albolabris; Daboia siamensis;
Deinagkistrodon acutus; Gloydius brevicaudus; Protobothrops
mucrosquamatus
Cat 2: Colubridae: Rhabdophis tigrinus; Elapidae: Bungarus bungaroides (south-east
Tibet), Bungarus fasciatus; Naja kaouthia; Ophiophagus hannah;
Viperidae: Trimeresurus septentrionalis (south Tibet); Gloydius halys,
Gloydius intermedius, Gloydius ussuriensis; Himalayophis tibetanus
(south Tibet); Protobothrops jerdonii, Protobothrops kaulbacki,
Protobothrops mangshanensis; Vipera berus (Jilin, western Xinjiang),
Vipera renardi (western Xinjiang); Trimeresurus stejnegeri
Taiwan Province
Cat 1: Elapidae: Bungarus multicinctus; Naja atra;
Viperidae: Protobothrops mucrosquamatus; Trimeresurus stejnegeri
Cat 2: Viperidae: Deinagkistrodon acutus; Daboia siamensis
Republic of Korea:
Cat 1: Viperidae: Gloydius brevicaudus
Cat 2: Colubridae: Rhabdophis tigrinus; Viperidae: Gloydius intermedius,
Gloydius ussuriensis
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South Asia
Afghanistan:
Cat 1: Elapidae: Naja oxiana; Viperidae: Echis carinatus; Macrovipera lebetina
Cat 2: Elapidae: Bungarus caeruleus (east), Bungarus sindanus (east); Naja naja (poss.
south-east); Viperidae: Eristicophis macmahonii (south-west); Gloydius halys
(north)
Bangladesh:
Cat 1: Elapidae: Bungarus caeruleus, Bungarus niger, Bungarus walli; Naja kaouthia;
Viperidae: Trimeresurus erythrurus
Cat 2: Elapidae: Bungarus bungaroides, Bungarus fasciatus, Bungarus lividus;
Naja naja; Ophiophagus hannah; Viperidae: Trimeresurus albolabris (far north-
west); Daboia russelii (west)
Bhutan:
Cat 1: Elapidae: Bungarus niger; Naja naja
Cat 2: Elapidae: Bungarus caeruleus, Bungarus fasciatus, Bungarus lividus;
Naja kaouthia; Ophiophagus hannah; Viperidae: Trimeresurus erythrurus;
Daboia russelii; Protobothrops jerdonii
India:
Cat 1: Elapidae: Bungarus caeruleus; Naja kaouthia (east), Naja naja (throughout);
Viperidae: Daboia russelii; Echis carinatus; Hypnale hypnale (south-west)
Cat 2: Bungarus bungaroides, Bungarus fasciatus, Bungarus lividus, Bungarus niger,
Bungarus sindanus, Bungarus walli; Naja oxiana (west), Naja sagittifera
(Andaman Islands); Ophiophagus hannah (south, north-east, Andaman
Islands); Viperidae: Trimeresurus albolabris, Trimeresurus erythrurus,
Trimeresurus septentrionalis; Gloydius himalayanus; Protobothrops jerdonii,
Protobothrops kaulbacki, Protobothrops mucrosquamatus;
Trimeresurus gramineus (south India), Trimeresurus malabaricus (south-west),
Nepal:
Cat 1: Elapidae: Bungarus caeruleus, Bungarus niger; Naja naja, Naja kaouthia;
Viperidae: Daboia russelii
Cat 2: Elapidae: Bungarus bungaroides, Bungarus fasciatus, Bungarus lividus,
Bungarus walli; Ophiophagus hannah; Viperidae: Trimeresurus septentrionalis;
Gloydius himalayanus; Himalayophis tibetanus; Protobothrops jerdonii
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Pakistan:
Cat 1: Elapidae: Bungarus caeruleus, Bungarus sindanus; Naja naja, Naja oxiana;
Viperidae: Daboia russelii; Echis carinatus
Cat 2: Viperidae: Eristicophis macmahonii (west); Gloydius himalayanus (north);
Macrovipera lebetina (west)
Sri Lanka:
Cat 1: Elapidae: Bungarus caeruleus; Naja naja; Viperidae: Daboia russelii;
Hypnale hypnale
Cat 2: Elapidae: Bungarus ceylonicus; Viperidae: Echis carinatus; Hypnale nepa,
Hypnale zara; Trimeresurus trigonocephalus
South-East Asia
Brunei Darussalam:
Cat 1: Elapidae: Naja sumatrana
Cat 2: Elapidae: Bungarus fasciatus, Bungarus flaviceps; Calliophis bivirgatus,
Calliophis intestinalis; Ophiophagus hannah;
Viperidae: Trimeresurus sumatranus; Tropidolaemus subannulatus
Cambodia:
Cat 1: Elapidae: Bungarus candidus; Naja kaouthia, Naja siamensis;
Viperidae: Calloselasma rhodostoma; Trimeresurus albolabris;
Daboia siamensis
Cat 2: Elapidae: Bungarus fasciatus, Bungarus flaviceps; Ophiophagus hannah;
Viperidae: Trimeresurus cardamomensis
Cat 1: Elapidae: Bungarus candidus (Sumatra and Java); Naja sputatrix (Java and
Lesser Sunda Islands), Naja sumatrana (Sumatra and Borneo);
Viperidae: Calloselasma rhodostoma (Java); Trimeresurus albolabris;
Daboia siamensis
Cat 2: Elapidae: Bungarus fasciatus, Bungarus flaviceps (Sumatra and Borneo);
Calliophis bivirgatus, Calliophis intestinalis; Ophiophagus hannah
(Sumatra, Borneo & Java); Viperidae: Trimeresurus insularis,
Trimeresurus purpureomaculatus (Sumatra), Trimeresurus sumatranus;
Tropidolaemus subannulatus
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Malaysia:
Cat 1: Elapidae: Bungarus candidus (Peninsular Malaysia); Naja kaouthia (northern
Peninsular Malaysia), Naja sumatrana (Peninsular Malaysia, Sabah and
Sarawak); Viperidae: Calloselasma rhodostoma
Cat 2: Elapidae: Bungarus fasciatus, Bungarus flaviceps; Calliophis bivirgatus,
Calliophis intestinalis; Ophiophagus hannah;
Viperidae: Trimeresurus purpureomaculatus, Trimeresurus hageni;
Tropidolaemus subannulatus
Myanmar:
Cat 1: Elapidae: Bungarus magnimaculatus, Bungarus multicinctus;
Naja kaouthia, Naja mandalayensis; Viperidae: Trimeresurus albolabris,
Trimeresurus erythrurus; Daboia siamensis
Cat 2: Elapidae: Bungarus bungaroides (Chin State), Bungarus candidus
(Thaninthayi Div.), Bungarus flaviceps (east Shan State), Bungarus niger;
Ophiophagus hannah; Viperidae: Calloselasma rhodostoma (Thaninthayi
Div.); Trimeresurus purpureomaculatus; Protobothrops jerdonii,
Protobothrops kaulbacki, Protobothrops mucrosquamatus (Kachin)
Philippines:
Cat 1: Elapidae: Naja philippinensis (Luzon), Naja samarensis (Mindanao),
Naja sumatrana (Palawan)
Cat 2: Elapidae: Calliophis intestinalis; Ophiophagus hannah;
Viperidae: Trimeresurus flavomaculatus; Tropidolaemus philippensis,
Tropidolaemus subannulatus
Singapore:
Cat 1: Elapidae: Bungarus candidus; Naja sumatrana
Cat 2: Elapidae: Bungarus fasciatus; Calliophis bivirgatus, Calliophis intestinalis;
Ophiopahgus hannah; Viperidae: Trimeresurus purpureomaculatus
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Thailand:
Cat 1: Elapidae: Bungarus candidus; Naja kaouthia, Naja siamensis;
Viperidae: Calloselasma rhodostoma; Trimeresurus albolabris;
Daboia siamensis
Cat 2: Elapidae: Bungarus fasciatus, Bungarus flaviceps; Calliophis bivirgatus,
Calliophis intestinalis; Naja sumatrana; Ophiophagus hannah;
Viperidae: Trimeresurus macrops, Trimeresurus hageni
Timor-Leste:
Cat 1: Viperidae: Trimeresurus insularis
Cat 2: None
Viet Nam:
Cat 1: Elapidae: Bungarus candidus, Bungarus multicinctus, Bungarus slowinskii
(north); Naja atra (north), Naja kaouthia (south);
Viperidae: Calloselasma rhodostoma; Trimeresurus albolabris (throughout)
Cat 2: Elapidae: Bungarus fasciatus, Bungarus flaviceps (south); Naja siamensis
(south); Ophiophagus hannah; Viperidae: Trimeresurus rubeus;
Protobothrops jerdonii, Protobothrops mucrosquamatus (north);
Trimeresurus stejnegeri; Deinagkistrodon acutus
Pseudechis australis is common and widespread and causes numerous snake-bites; bites may be severe,
6
although this species has not caused a fatality in Australia since 1968.
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EUROPE
There are no venomous snakes in Iceland, Ireland, Isle of Man, Outer Hebrides,
Orkney or the Shetland Islands. Crete and most of the islands of the western
Mediterranean are also free of venomous snakes.
Central Europe
Albania, Bulgaria, Romania, Serbia, Montenegro, Slovenia, The former Yugoslav
Republic of Macedonia:
Cat 1: Viperidae: Vipera ammodytes
Cat 2: Viperidae: Vipera berus
Croatia:
Cat 1: Viperidae: Vipera ammodytes
Cat 2: Viperidae: Vipera berus
Czechia:
Cat 1: None
Cat 2: Viperidae: Vipera berus
Greece:
Cat 1: Viperidae: Vipera ammodytes
Cat 2: Viperidae: Macrovipera schweizeri; Montivipera xanthina; Vipera berus
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Hungary:
Cat 1: None
Cat 2: Viperidae: Vipera berus
Poland:
Cat 1: None
Cat 2: Viperidae: Vipera berus
Slovakia:
Cat 1: None
Cat 2: Viperidae: Vipera berus
Eastern Europe
Belarus, Estonia, Latvia, Lithuania, Republic of Moldova:
Cat 1: None
Cat 2: Viperidae: Vipera berus, Vipera nikolskii (Moldova)
Russian Federation:
Cat 1: Viperidae: Vipera berus
Cat 2: Viperidae: Gloydius halys, Gloydius intermedius, Gloydius ussuriensis (far-east
Russia); Macrovipera lebetina (Dagestan); Vipera nikolskii, Vipera renardi,
Vipera spp.
Ukraine:
Cat 1: None
WHO Technical Report Series, No. 1004, 2017
Western Europe
Austria:
Cat 1: None
Cat 2: Viperidae: Vipera ammodytes, Vipera berus
Belgium:
Cat 1: None
Cat 2: Viperidae: Vipera berus
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Denmark:
Cat 1: None
Cat 2: Viperidae: Vipera berus
Finland:
Cat 1: None
Cat 2: Viperidae: Vipera berus
France:
Cat 1: Viperidae: Vipera aspis
Cat 2: Viperidae: Vipera berus
Germany:
Cat 1: None
Cat 2: Viperidae: Vipera berus
Italy:
Cat 1: Viperidae: Vipera aspis
Cat 2: Viperidae: Vipera ammodytes, Vipera berus
the Netherlands:
Cat 1: None
Cat 2: Viperidae: Vipera berus
Norway:
Cat 1: None
Cat 2: Viperidae: Vipera berus
Portugal:
Cat 1: None
Cat 2: Viperidae: Vipera latastei, Vipera seoanei
Spain:
Cat 1: None
Cat 2: Viperidae: Vipera aspis, Vipera latastei, Vipera seoanei
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Sweden:
Cat 1: Viperidae: Vipera berus
Cat 2: None
Switzerland:
Cat 1: None
Cat 2: Viperidae: Vipera aspis, Vipera berus
THE AMERICAS
North America
Canada:
Cat 1: None
Cat 2: Viperidae: Crotalus oreganus, Crotalus viridis; Sistrurus catenatus
Mexico:
Cat 1: Viperidae: Agkistrodon bilineatus, Agkistrodon taylori; Crotalus atrox,
Crotalus scutulatus, Crotalus simus, Crotalus molossus; Bothrops asper
Cat 2: Elapidae: Micruroides euryxanthus, Micrurus nigrocinctus, Micrurus tener,
Micrurus spp.; Viperidae: Agkistrodon contortrix, Agkistrodon russeolus;
Atropoides mexicanus, Atropoides occiduus, Atropoides spp.;
Bothriechis schlegelii, Bothriechis spp.; Cerrophidion godmani,
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Central America
The most medically important species are Crotalus simus and Bothrops asper.
Belize:
Cat 1: Viperidae: Bothrops asper
Cat 2: Elapidae: Micrurus spp.; Viperidae: Agkistrodon russeolus;
Atropoides mexicanus; Bothriechis schlegelii; Crotalus tzabcan;
Porthidium nasutum
Costa Rica:
Cat 1: Viperidae: Bothrops asper; Crotalus simus
Cat 2: Elapidae: Micrurus nigrocinctus, Micrurus spp.;
Viperidae: Agkistrodon howardgloydi; Atropoides mexicanus,
Atropoides picadoi; Bothriechis schlegelii, Bothriechis lateralis, Bothriechis spp.;
Cerrophidion sasai; Lachesis melanocephala, Lachesis stenophrys;
Porthidium nasutum, Porthidium ophrymegas, Porthidium spp.
El Salvador:
Cat 1: Viperidae: Crotalus simus
Cat 2: Elapidae: Micrurus nigrocinctus; Micrurus spp.;
Viperidae: Agkistrodon bilineatus; Atropoides occiduus; Bothriechis spp.;
Cerrophidion wilsoni; Porthidium ophryomegas
Guatemala:
Cat 1: Viperidae: Bothrops asper; Crotalus simus
Cat 2: Elapidae: Micrurus nigrocinctus, Micrurus spp.;
Viperidae: Agkistrodon bilineatus, Agkistrodon russeolus; Atropoides mexicanus,
Atropoides occiduus; Bothriechis schlegelii, Bothriechis spp.;
Cerrophidion godmani; Crotalus tzabcan, Porthidium nasutum,
Porthidium ophryomegas
Honduras:
Cat 1: Viperidae: Bothrops asper
Cat 2: Elapidae: Micrurus nigrocinctus, Micrurus spp.;
Viperidae: Agkistrodon howardgloydi; Atropoides mexicanus, Atropoides spp.;
Bothriechis marchi, Bothriechis schlegelii, Bothriechis spp.; Cerrophidion wilsoni;
Crotalus simus; Porthidium nasutum, Porthidium ophryomegas
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Nicaragua:
Cat 1: Viperidae: Bothrops asper; Crotalus simus
Cat 2: Elapidae: Micrurus nigrocinctus, Micrurus spp.;
Viperidae: Agkistrodon howardgloydi; Atropoides mexicanus;
Bothriechis schlegelii; Cerrophidion godmani; Lachesis stenophrys;
Porthidium nasutum, Porthidium ophryomegas
Panama:
Cat 1: Viperidae: Bothrops asper
Cat 2: Elapidae: Micrurus mipartitus, Micrurus nigrocinctus, Micrurus spp.;
Viperidae: Atropoides mexicanus, Atropoides spp.; Bothriechis lateralis,
Bothriechis schlegelii, Bothriechis spp.; Cerrophidion sasai; Lachesis acrochorda,
Lachesis stenophrys; Porthidium nasutum, Porthidium lansbergii,
Porthidium spp.
Caribbean
No medically important snakes occur naturally in Anguilla, Antigua and Barbuda,
the Bahamas, Barbados, Bermuda, The British Virgin Islands, Cayman Islands,
Cuba, Dominica, the Dominican Republic, Grenada, Guadeloupe, Haiti, Jamaica,
Montserrat, the Netherlands Antilles, Saint Kitts and Nevis, Saint Vincent and
the Grenadines, and Turks and Caicos Islands.
Aruba, Martinique, Saint Lucia, Trinidad and Tobago, and offshore islands:
Cat 1: Viperidae: Bothrops cf. atrox (Trinidad), Bothrops caribbaeus (St Lucia),
Bothrops lanceolatus (Martinique); Crotalus durissus (Aruba)
Cat 2: Elapidae: Micrurus circinalis (Trinidad), Micrurus lemniscatus (Trinidad);
Viperidae: Lachesis muta (Trinidad)
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South America
No venomous snakes are occur naturally in the Falkland Islands and no
dangerously venomous snakes occur naturally in Chile.
Argentina:
Cat 1: Viperidae: Bothrops alternatus, Bothrops diporus; Crotalus durissus
Cat 2: Elapidae: Micrurus corallinus, Micrurus lemniscatus, Micrurus spp.;
Viperidae: Bothrops ammodytoides, Bothrops jararaca, Bothrops jararacussu,
Bothrops mattogrossensis, Bothrops neuwiedi, Bothrops pubescens
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Brazil:
Cat 1: Viperidae: Bothrops atrox, Bothrops jararaca, Bothrops jararacussu,
Bothrops leucurus, Bothrops moojeni; Crotalus durissus
Cat 2: Elapidae: Micrurus corallinus, Micrurus lemniscatus, Micrurus spixii,
Micrurus surinamensis, Micrurus spp.; Viperidae: Bothrocophias hyoprora;
Bothrocophias microphthalmus; Bothrops alternatus, Bothrops bilineatus,
Bothrops brazili, Bothrops diporus, Bothrops mattogrossensis,
Bothrops neuwiedi, Bothrops pubescens, Bothrops taeniatus, Bothrops spp.;
Lachesis muta
Colombia:
Cat 1: Viperidae: Bothrops asper, Bothrops atrox, Bothrops bilineatus;
Crotalus durissus
Cat 2: Elapidae: Micrurus lemniscatus, Micrurus mipartitus, Micrurus nigrocinctus,
Micrurus spixii, Micrurus surinamensis, Micrurus spp.;
Viperidae: Bothriechis schlegelii; Bothrocophias hyoprora,
Bothrocophias microphthalmus, Bothrocophias spp.; Bothrops brazili,
Bothrops taeniatus, Bothrops spp.; Lachesis acrochorda, Lachesis muta;
Porthidium nasutum, Porthidium lansbergii
Ecuador:
Cat 1: Viperidae: Bothrops asper, Bothrops atrox, Bothrops bilineatus;
Lachesis muta
Cat 2: Elapidae: Micrurus lemniscatus, Micrurus mipartitus, Micrurus spixii,
Micrurus surinamensis, Micrurus spp.; Viperidae: Bothriechis schlegelii;
Bothrocophias hyoprora, Bothrocophias microphthalmus, Bothrocophias spp.;
Bothrops brazili, Bothrops taeniatus, Bothrops spp.; Lachesis acrochorda;
Porthidium nasutum, Porthidium spp.
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Guyana:
Cat 1: Viperidae: Bothrops atrox, Bothrops bilineatus; Crotalus durissus
Cat 2: Elapidae: Micrurus lemniscatus, Micrurus surinamensis, Micrurus spp.;
Viperidae: Bothrops brazili, Bothrops taeniatus; Lachesis muta
Paraguay:
Cat 1: Viperidae: Bothrops alternatus; Crotalus durissus
Cat 2: Elapidae: Micrurus corallinus, Micrurus lemniscatus, Micrurus spixii,
Micrurus spp.; Viperidae: Bothrops diporus, Bothrops jararaca,
Bothrops jararacussu, Bothrops mattogrossensis, Bothrops moojeni,
Bothrops neuwiedi, Bothrops spp.
Peru:
Cat 1: Viperidae: Bothrops atrox, Bothrops bilineatus, Bothrops pictus;
Crotalus durissus; Lachesis muta
Cat 2: Elapidae: Micrurus lemniscatus, Micrurus mipartitus, Micrurus spixii,
Micrurus surinamensis, Micrurus spp.; Viperidae: Bothriechis schlegelii;
Bothrocophias hyoprora, Bothrocophias microphthalmus; Bothrops asper;
Bothrops brazili, Bothrops mattogrossensis, Bothrops taeniatus, Bothrops spp.
Suriname:
Cat 1: Viperidae: Bothrops atrox, Bothrops bilineatus; Crotalus durissus
Cat 2: Elapidae: Micrurus lemniscatus, Micrurus surinamensis, Micrurus spp.;
Viperidae: Bothrops brazili, Bothrops taeniatus; Lachesis muta
Uruguay:
Cat 1: Viperidae: Bothrops alternatus; Bothrops pubescens
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Annex 5
Herpetological references 7
Abtin E, Nilson G, Mobaraki A, Hosseini AA, Dehgannejhad M. A new species of Krait, Bungarus (Reptilia,
Elapidae, Bungarinae) and the first record of that genus in Iran. Russ J Herpetol. 2014;21(4):243–50.
Ananjeva NB, Orlov NL, Khalikov RG, Darevsky IS, Ryabov IS et al. The reptiles of Northern Eurasia. Sofia:
Pensoft; 2006.
Anderson CG, Greenbaum E. Phylogeography of northern populations of the Black-Tailed Rattlesnake
(Crotalus molossus Baird and Girard, 1853), with the revalidation of C. ornatus Hallowell, 1854. Herpetol
Monogr. 2012;26(1):19–57.
Ashton KG, de Querioz A. Molecular systematics of the western rattlesnake, Crotalus viridis (Viperidae),
with comments on the utility of the D-loop in phylogenetic studies of snakes. Mol Phylogenet Evol.
2001;21:176–89.
Babocsay G. A new species of saw-scaled viper of the Echis coloratus complex (Ophidia: Viperidae) from
Oman, Eastern Arabia. Syst Biodivers. 2004;1:503–14.
Basoglu M, Baran I. The reptiles of Turkey. Part II. The snakes. Izmir: University of Matbaasi; 1980.
Buys PJ, Buys PJC. Snakes of Namibia. Windhoek; Gamsberg Macmillan; 1983.
Bons J, Geniez P. Amphibiens et reptiles du Maroc. Barcelona; Asociación Herpetológica Española; 1996.
Boycott RC. A herpetological survey of Swaziland [MSc thesis]. Durban: University of Natal; 1992.
Branch B. Field guide to the snakes and other reptiles of Southern Africa. Cape Town: Sruik; 1988.
Broadley DG, Doria CT, Wigge J. Snakes of Zambia: An atlas and field guide. Frankfurt am Main: Edition
Chimair; 2003.
Broadley DG. FitzSimons snakes of Southern Africa. Cape Town: Delta Books; 1983.
Broadley DG. The herpetofaunas of the islands off the coast of south Moçambique. Arnoldia Zimbabwe.
1990;9:469–93.
Broadley DG. Reptiles and amphibians from the Bazaruto Archipelago, Mozambique. Arnoldia
Zimbabwe. 1992;9:539–48.
Broadley DG. Review of the Dispholidini, with the description of a new genus and species from Tanzania
(Serpentes, Colubridae). Bull Br Mus Nat Hist Zool. 2002;68:57–74.
Broadley DG. A review of the genus Thelotornis A. Smith in eastern Africa, with the description of a
new species from the Usambara Mountains (Serpentes: Colubridae: Dispholidini). Afr J Herpetol.
2001;50:53–70.
Broadley DG. The herpetofaunas of the islands off the coast of south Moçambique. Arnoldia Zimbabwe.
1990;9:469–493.
Campbell JA, Lamar WW. The venomous snakes of the western hemisphere. Vols. I and II. Ithaca, NY:
Comstock-Cornell; 2004.
Creer S, Malhotra A, Thorpe RS, Stöcklin RS, Favreau PS, Hao Chou WS. Genetic and ecological
correlates of intraspecific variation in pitviper venom composition detected using matrix-assisted
laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and isoelectric focusing. J Mol
Evol. 2003;56:317–29.
da Silva VX. The Bothrops neuwiedi complex. In: Campbell JA, Lamar WW, editors. The venomous
reptiles of the Western Hemisphere. Vol. I. Ithaca (NY): Comstock Publishing; 2004:410–22.
381
WHO Expert Committee on Biological Standardization Sixty-seventh report
David P, Ineich I. Les serpents venimeux du monde: systématique et répartition. Dumerilia. 1999;
3:3–499.
David P, Vogel G, Dubois A. On the need to follow rigorously the Rules of the Code for the subsequent
designation of a nucleospecies (type species) for a nominal genus which lacked one: the case of the
nominal genus Trimeresurus Lacépède, 1804 (Reptilia: Squamata: Viperidae). Zootaxa. 2011;2992:1–51.
De Smedt J. The vipers of Europe. Hablech: JDS Verlag; 2001.
Dobiey M, Vogel G. Venomous snakes of Africa. Frankfurt am Main: Terralog Edition Chimaira; 2006.
El Din SB. A guide to the reptiles and amphibians of Egypt. Cairo: American University of Cairo Press;
2006.
Gasperetti J. Snakes of Arabia. Fauna Saudi Arabia. 1988;9:169–450.
Geniez P, Mateo A, Geniez M, Pether J. The amphibians and reptiles of the Western Sahara. Frankfurt am
Main: Edition Chimaira; 2004.
Gloyd HK, Conant R. Snakes of the Agkistrodon complex. Oxford: OH, SSAR; 1990.
Gower DJ, Wade EOZ, Spaels S, Böhme W, Buechley ER, Sykes D, Colston TJ. A new large species of Bitis
Gray, 1842 (Serpentes: Viperidae) from the Bale Mountains of Ethiopia. Zootaxa. 2016;4093(1):41–63.
Guo P, Malhotra A, Li PP, Creer S. New evidence on the phylogenetic position of the poorly known Asian
pitviper Protobothrops kaulbacki (Serpentes: Viperidae: Crotalinae) with a redescription of the species
and a revision of the genus Protobothrops. Herpetol J. 2007;17:237–46.
Gumprecht A et al. Asian Pitvipers. Berlin: Geitje Books; 2004.
Gutberlet RL Jr, Campbell JA. Generic recognition of a neglected lineage of South American pitvipers
(Squamata: Viperidae: Crotalinae), with the description of a new species from the Colombian Chocó. Am
Museum Novitates. 2001;3316:1–15.
Jadin, RC, Townsend, JH, Castoe TA, Campbell JA. Cryptic diversity in disjunct populations of Middle
American Montane Pitvipers: a systematic reassessment of Cerrophidion godmani. Zool Scripta.
2012;41:455–70.
Kästle W, Rai K, Schleich H. Amphibians and reptiles of Nepal. Ruggall: Gantner Verlag; 2002.
Khan MS. A guide to the snakes of Pakistan. Khan MS. A guide to the snakes of Pakistan. Frankfurt am
Main: Edition Chimaira; 2002.
Kreiner G. The snakes of Europe. Frankfurt am Main: Edition Chimaira; 2007.
Latifi M. The snakes of Iran. Oxford: OH:SSAR; 1985.
WHO Technical Report Series, No. 1004, 2017
Malhotra A, Thorpe RS. A phylogeny of four mitochondrial gene regions suggests a revised taxonomy
for Asian pitvipers (Trimeresurus and Ovophis). Mol Phylogenet Evol. 2004;32:83–100.
McDiarmid RW, Campbell JA, Toure T. Snake species of the world: A taxonomic and geographical
reference. Vol. 1. Wahington, DC: Herpetologists’ League: 1999.
Murphy JC. Amphibians and Reptiles of Trinidad and Tobago. Malabar, FL: Krieger; 1997.
Nilson G, Rastegar-Pouyani N. Walterinnesia aegyptia Lataste, 1887 (Ophidia: Elapidae) and the status of
Naja morgani Mocquard 1905. Russ J Herpetol. 2007;14:7–14.
Orlov NL, Barabanov AV. Analysis of nomenclature, classification, and distribution of the Agkistrodon
halys – intermedius complexes: a critical review. Russ J Herpetol. 1999;6:167–92.
O’Shea M. A guide to the snakes of Papua New Guinea. Port Moresby: Independent Publishing; 1996.
Parkinson CL Zamudio KR, Greene HW. Phylogeography of the pitviper clade Agkistrodon: historical
ecology, species status, and conservation of cantils. Mol Ecol. 2000;9:411–20.
Pitman CRS. A guide to the snakes of Uganda, second edition. Codicote, Wheldon & Wesley; 1974.
Porras LW, Wilson LD, Schuett GW, Reiserer R.S. A taxonomic reevaluation and conservation assessment
of the common cantil, Agkistrodon bilineatus (Squamata: Viperidae): a race against time. Amphib
Reptile Conserv. 2013;7(1):48–73.
Pyron RA, Burbrink FT, Colli GR, de Oca AN, Vitt LJ, Kuczynski CA et al. The phylogeny of advanced snakes
(Colubroidea), with discovery of a new subfamily and comparison of support methods for likelihood
trees. Mol Phylogenet Evol. 2011;58(2):329–42. doi:10.1016/j.ympev.2010.11.006.
Pyron RA, Dushantha Kandambi HK, Hendry CR, Pushpamal V, Burbrink FT, Somaweera R. Genus-
level phylogeny of snakes reveals the origins of species richness in Sri Lanka. Mol Phylogenet Evol.
2013;66(3):969–78
Pyron RA, Burbrink FT, Wiens JJ. A phylogeny and revised classification of Squamata, including 4161
species of lizards and snakes. BMC Evol Biol. 2013;13:93.
Quijada-Mascareñas JA Ferguson JE, Pook CE, Da Graca Saloma M, Thorpe RS, Wuester W. Phylogeographic
patterns of trans-Amazonian vicariants and Amazonian biogeography: the Neotropical rattlesnake
(Crotalus durissus complex) as an example. J Biogeogr 2007;34:1296–312.
Roman B. Serpents de Haute-Volta. Ouagadougou: CNRST; 1980.
Schleich HH, Kastle W, Kabish K. Amphibians and reptiles of North Africa. Koenigstein: Koeltz; 1996.
Spawls S, Branch B. The dangerous snakes of Africa. London: Blandford; 1995.
Spawls S, Howell A, Drewes R. A field guide to the reptiles of East Africa: Kenya, Tanzania, Uganda,
Rwanda and Burundi. London: Academic Press, Natural World; 2002.
Steward JW. The snakes of Europe. Newton Abbott: David & Charles; 1971.
Street D. Reptiles of northern and central Europe. London: Batsford; 1979.
Sweeney RCH. Snakes of Nyasaland. Amsterdam: Asher & Co; 1971.
Thorpe RS, Pook CE, Malhotra A. Phylogeography of the Russell’s viper (Daboia russelii) complex in
relation to variation in the colour pattern and symptoms of envenoming. Herpetol J. 2007;17:209–18.
Visser J, Chapman DS. Snakes and snakebite. Cape Town: Struik; 1978.
Vogel G. Venomous snakes of Asia. Frankfurt am Main: Terralog Edition Chimaira; 2006.
Vogel G, David P, Lutz M, van Rooijen, Vidal N. Revision of the Tropidolaemus wagleri-complex
(Serpentes: Viperidae: Crotalinae). I. Definition of included taxa and redescription of Tropidolaemus
wagleri (Boie, 1827). Zootaxa. 2007;1644:1–40.
Wallach V, Williams KL, Boundy J. Snakes of the world: A catalogue of living and extinct species. CRC
Press: London; 2014.
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Whitaker R, Captain A. Snakes of India: The field guide. Chennai: Draco Books; 2004.
Williams DJ, O’Shea M, Daguerre RL. Origin of the eastern brownsnake, Pseudonaja textilis (Duméril,
Bibron and Duméril) (Serpentes: Elapidae: Hydrophiinae) in New Guinea: evidence of multiple
dispersals from Australia, and comments on the status of Pseudonaja textilis pughi Hoser 2003.
Zootaxa. 2008;1703:47–61.
Williams DJ, Wüster W. Snakes of Papua New Guinea. In: Willliams DJ et al., editors. Venomous bites and
stings in Papua New Guinea. Melbourne: AVRU University of Melbourne; 2005:33–64.
Wüster W et al. Snakes across the strait: trans-Torresian phylogenetic relationships in three genera
of Australo-Papuan snakes (Serpentes: Elapidae: Acanthophis, Oxyuranus and Pseudechis). Molecular
Phylogenetics and Evolution, 2005, 34:1–14.
Wuster W, Broadley DG. A new species of spitting cobra from north-eastern Africa (Serpentes: Elapidae:
Naja). J Zool London. 2003;259:345–59.
Wüster W, Broadley DG. Get an eyeful of this: a new species of giant spitting cobra from eastern and
north-eastern Africa (Squamata: Serpentes: Elapidae: Naja). Zootaxa. 2007;1532:51–68.
Wuster W, Wüster W, Salomao MG, Quijada-Mascarenas JA, Thorpe RS. Origin and evolution of the
South American pitviper fauna: evidence from mitochondrial DNA sequence analysis. In: Schuett
GW, Höggren M, Douglas ME, Greene HW, editors. Biology of the vipers. Eagle Mountain, Utah: Eagle
Mountain Publishing; 2002:111–128.
Wüster W, Thorpe RS, da Graça Salomão M, Thomas L. Origin and phylogenetic position of the Lesser
Antillean species of Bothrops (Serpentes: Viperidae): biogeographical and medical implications. Bull.
Nat. Hist. Mus. London, Zool. 2002;68:101–6.
Wüster W, Warrell DA, Cox MJ, Jintakune P, Nabhitabhata J. Redescription of Naja siamensis Laurenti,
(Serpentes: Elapidae), a widely overlooked spitting cobra from Southeast Asia: geographic variation,
medical importance and designation of a neotype. J Zool, 1997; 243:771–88.
Wüster W Salomão MG, Thorpe RS, Puorto G, Furtado MFD, Hoge SA, et al. Systematics of the
Bothrops atrox species complex: insights from multivariate analysis and mitochondrial DNA sequence
information. In: Thorpe RS, Wüster W, Malhotra A. editors. Venomous snakes: ecology, evolution and
snakebite. Oxford: Clarendon Press; 1997:99–113 (Symposia of the Zoological Society of London,
No. 70).
Wüster W, Crookes S, Ineich I, Mane Y, Pook CE, Trape J-F et al. The phylogeny of cobras inferred from
mitochondrial DNA sequences: evolution of venom spitting and the phylogeography of the African
WHO Technical Report Series, No. 1004, 2017
spitting cobras (Serpentes: Elapidae: Naja nigricollis complex). Mol Phylogenet Evol. 2007;45:437–53.
Wüster W, Golay P, Warrell DA. Synopsis of recent developments in venomous snake systematics.
Toxicon. 1997;35:319–40.
Wüster W, Golay P, Warrell DA. Synopsis of recent developments in venomous snake systematics, No. 2.
Toxicon. 1998;36:299–307.
Wüster W, Golay P, Warrell DA. Synopsis of recent developments in venomous snake systematics, No. 3.
Toxicon. 1999;37:1123–29.
Wüster W, McCarthy CJ. Venomous snake systematics: Implications for snake bite treatment and
toxinology. In: Bon C, Goyffon M, editors. Envenomings and their treatments. Lyon: Fondation Mérieux;
1996:13–23.
Zhao E, Adler K. Herpetology of China. Oxford: OH, SSAR; 1993.
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Appendix 2
Model protocol for the production and testing of snake
antivenom immunoglobulins
Identification of the lot
Name and address of manufacturer
Determination of pH
–– Result
Concentration of preservatives (if used)
–– Type
–– Method
–– Result
Chemical agents used in plasma fractionation
–– Type
–– Method
–– Result
Inspection of final containers
–– Results
Residual moisture in freeze-dried antivenoms
–– Method
–– Result
Internal certification
Certification by person taking overall responsibility for production of the antivenom
I certify that batch no. of
snake antivenom immunoglobulin meets all national requirements and/or
satisfies the 2016 WHO Guidelines for the production, control and regulation
of snake antivenom immunoglobulins.1
Signature
Name (typed)
Date
WHO Technical Report Series, No. 1004, 2017
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WHO manual for the preparation of secondary reference
materials for in vitro diagnostic assays designed for
infectious disease nucleic acid or antigen detection:
calibration to WHO International Standards
1. Background 392
2. Purpose and scope 392
3. Terminology 393
4. Principles of biological standardization 395
5. Calibration hierarchy of biological standards 395
5.1 WHO International Standards 398
5.2 Secondary standards 399
5.3 Tertiary standards 400
5.4 Other control material 400
6. Commutability of biological standards 400
7. Selection and characterization of materials for the preparation
of secondary standards – and application to tertiary standards 401
7.1 Analyte – biological versus synthetic material 401
7.2 Immunological and genetic diversity 402
7.3 Matrix 403
7.4 Concentration 403
7.5 Volume 403
7.6 Diagnostic specificity 404
7.7 Infectivity 404
7.8 Physical appearance 404
7.9 Homogeneity 405
7.10 Stability 405
7.11 Stability assessment during product lifetime 406
7.12 In-use stability 407
8. Calibration: testing and statistical analysis 408
8.1 Principles of calibration 408
8.2 Single assay calibration using qualitative tests 409
8.3 Single assay calibration using quantitative tests 410
8.4 Collaborative study calibration using multiple assays 412
8.5 Calculation of uncertainty of measurement 412
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Abbreviations
Ag antigen
% CV percentage coefficient of variation
Ct cycle threshold
DNA deoxyribonucleic acid
EQA external quality assurance
IS International Standard(s)
IU International Unit(s)
IVD in vitro diagnostic
MU measurement uncertainty
NAT nucleic acid amplification technique
NRA national regulatory authority
PT proficiency testing
RNA ribonucleic acid
S/CO sample-to-cut-off (ratio); also signal-to-cut-off (ratio)
Système international d’unités (measurement system using
SI
metric units)
SoGAT Standardisation of Genome Amplification Techniques (group)
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1. Background
Through its Expert Committee on Biological Standardization, WHO developed
its Guidelines for the preparation and establishment of reference materials and
reference reagents for biological substances in 1978 (1). This document was
last revised in 2004 (2). In the revised WHO Recommendations document,
secondary standards are defined as reference preparations established by regional
or national authorities, or by other laboratories, that are calibrated against and
traceable to WHO reference materials. Part B of the 2004 Recommendations deals
with general considerations for the preparation, characterization and calibration
of regional or national biological reference standards.
Feedback from manufacturers and providers of secondary (for example,
regional) standards used for in vitro diagnostic (IVD) devices, and from
regulatory authorities, international trade organizations, IVD manufacturers,
providers of external quality assurance (EQA) or proficiency testing (PT)
programmes and laboratories using diagnostic assays, indicated a need for more
specific guidance on the preparation of secondary standards; it was therefore
concluded that a practical manual focusing on IVD needs would be helpful.
This topic was discussed at the 2012 meeting of the WHO Expert Committee on
Biological Standardization, at which the proposal to generate a WHO document
on secondary standards for use in the IVD field was endorsed.
of infectious diseases where the typical analytes (measurands) are nucleic acid
or antigen (Ag). These IVD tests cover nucleic acid amplification technique
(NAT)-based assays for detecting the DNA or RNA of infectious agents and
immunological tests for the detection of Ag(s) of infectious agents. Currently,
there are only a small number of IS with an assigned unitage available where
the analyte is an antibody directed to an infectious agent. Due to their complexity
(that is, the epitope spectrum represented by polyclonal antibodies in the
serum of a patient) this document does not cover antibody-based secondary
standards. However, several principles outlined in this manual may also apply to
antibody assays. Where applicable, the document integrates existing guidance,
referenced accordingly.
The document is intended for use by manufacturers of secondary
reference materials, IVD manufacturers, providers of EQA or PT programmes
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and other laboratories using reference materials for NAT-based and serological
infectious disease assays. Analogous guidance has already been issued by WHO
on secondary standards for vaccines (3) and chemical reference substances (4).
3. Terminology
The definitions given below apply to the terms as used in this WHO guidance
document. These terms may have different meanings in other contexts.
Accuracy: (measurement) closeness of agreement between a measured
quantity value and the true quantity value of a measurand (5).
Biological matrix: a discrete material of biological origin that can be
sampled and processed in a reproducible manner. Examples include blood,
serum, plasma, urine, faeces, saliva, sputum and various discrete tissues (6).
Calibration: a process that, under specified conditions, establishes as a
first step a relation between the quantity values with measurement uncertainties
provided by measurement standards and corresponding indications with
associated measurement uncertainties and, in a second step, uses this information
to establish a relation for obtaining a measurement result from an indication (5).
Calibration hierarchy: a sequence of calibrations from a reference to the
final measuring system, where the outcome of each calibration depends on
the outcome of the previous calibration (5).
Commutability (of a reference material): a property of a reference
material, demonstrated by the equivalence of the mathematical relationships
among the results of different measurement procedures for a reference material
and for representative samples of the type intended to be measured (7).
Control material: a substance, material or article used to verify the
performance characteristics of an in vitro diagnostic (IVD) medical device (8).
Diagnostic specificity: the probability that the device gives a negative
result in the absence of the target marker (9).
End-point titre: the reciprocal of the highest analyte dilution that gives a
reading above the assay cut-off (10).
International measurement standard: a measurement standard
recognized by signatories to an international agreement and intended to serve
worldwide, for example a WHO International Standard (IS) (5).
International conventional calibrator: a calibrator whose value of a
quantity is not metrologically traceable to SI units but is assigned by international
agreement (11).
International Unit(s) (IU): the unitage assigned by WHO to an
International Biological Standard (2).
Linearity (of a measuring system): the ability to provide measured
quantity values that are directly proportional to the value of the measurand in
the sample (12).
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of prefixes and their names and symbols), together with rules for their use,
adopted by the General Conference on Weights and Measures (5).
Traceability: (metrological) property of a measurement result whereby
the result can be related to a reference through a documented unbroken chain
of calibrations, each contributing to the measurement uncertainty (5).
Threshold cycle: the polymerase chain reaction (PCR) cycle at which
the gain in fluorescence generated by the accumulating amplicon exceeds a
threshold over baseline – for example, defined as 10 standard deviations of the
mean baseline fluorescence using data taken from cycles 3 to 15 (15).
Working standard: a measurement standard used routinely to calibrate
or verify measuring instruments or measuring systems (5).
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Fig. A6.1
Calibration hierarchy and metrological traceability to the WHO IS
Uncertainty of measurement
Traceability
Table A6.1
Key properties of WHO IS, secondary standards and tertiary standards
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As with all biological references, the material used should resemble as closely as
possible the natural analyte of the clinical sample to be measured. An assessment
of commutability should be performed as part of the collaborative study where
appropriate and feasible (2).
By definition, an IS has a specified value expressed in International Units
(IU). This value is arbitrarily assigned based on the results of the collaborative
study. The assigned IU value of each IS (new and replacement) does not carry an
uncertainty associated with calibration (see section 8.1 below). The uncertainty
is considered to be the variance of the vial weight determined during the filling
process (20, 21). The collaborative study design attempts to ensure continuity
of the IU as far as possible. As explained further in section 8.1 there is no
uncertainty value associated with replacements.
As the highest-order standard for biological material, the use of a
WHO IS should be limited to the calibration of secondary biological reference
materials in order to minimize the need to replace the IS on a regular basis.
Unfortunately, the limited availability of secondary calibrator standards and the
lack of specific guidance on the establishment and calibration of more readily
available standards have resulted in the overuse of WHO IS for more routine
procedures such as validation of assays and as run controls.
Biological stock materials assigned a potency based on calibration against the IS by using exclusively
1
one specific test, and used, for example, for the preparation of calibrators or run controls for this test,
are also considered as in-house secondary standards. Non-biological preparations such as synthetic
preparations (for example, plasmid preparations, transcripts, armored RNA and antigens produced by
recombinant DNA technology) are often used for test calibration. If this calibration is done against an
IS, these materials will also be considered as secondary standards under the scope of this document.
Nevertheless, these materials have a number of limitations compared to the biological preparations, for
example, in terms of commutability.
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demonstrating commutability for two assay methods does not guarantee that
there will be commutability with other methods. Similarly, if a set of samples
demonstrate commutability with each other for particular assay methods then
this does not guarantee that this will apply to all samples (22, 23).
However, ISO17511:2003 states that calibrators are to be commutable
at each step in the traceability chain (11). There are established recommendations
for the assessment of commutability of reference materials used in laboratory
medicine (7).
Ideally, the material selected should resemble the analyte in the IS and in
usual clinical specimens as closely as possible. The decision may be based on
the availability of a sufficient volume of the material to enable preparation of
a single batch of secondary standard that, when frozen, will last several years.
In most cases, laboratory strains of microorganisms are better characterized
and available in larger volumes than clinical strains. However, the latter may
better represent the samples that are routinely tested. Where a whole organism
is unavailable in sufficient quantities, a laboratory-derived material (such as
a purified nucleic acid preparation) may be the only option. Demonstrating
the commutability of such a material to different clinical samples may be
challenging. This will need to be addressed on a case-by-case basis and should
be done in cases where, through experimental assessment, it is proven that
the use of laboratory-derived material improves agreement between assays.
Analytes derived directly from human origin (such as a clinical sample) or
the matrix of a biological sample (such as plasma or whole blood) should be
tested and confirmed to be negative for the presence of pathogens other than
the analyte of interest. This should be done to exclude potential cross-reactivity
with the specific target analyte. If it is necessary to prepare a bulk material by
pooling from more than one source, each component of the bulk material must
be characterized and where possible all components should be identical – for
example, in molecular detection the sequence of the target regions should be
the same. All samples pooled must be mixed thoroughly and the pool should
be homogeneous. It should be noted that pooling may not be appropriate in
all circumstances. A biological bulk material with a high analyte concentration
could if needed be diluted in a suitable matrix.
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7.3 Matrix
The matrix in which the standard is formulated is crucial to creating a material
that is fit for purpose. For NAT-based assays, the matrix needs to be appropriate
for the assay or range of assays for which it is intended. Multiple sample types may
be considered. It follows that for commutability purposes, a biological matrix
would be preferred over a synthetic matrix. It should be taken into consideration
that some sample matrices include inhibitory factors that interfere with the
performance of specific types of assays (7). Furthermore, the chosen matrix of
the reference preparation should be compatible with further matrices into which
it may be spiked. For example, where a pathogen may be screened for in plasma,
whole blood or urine, it may not be appropriate to formulate a material in a
matrix that cannot be further diluted in clinical samples. In the case of plasma,
consideration should also be given to any anticoagulant treatment and to any
additional treatments such as cryoprecipitation or recalcification.
7.4 Concentration
Secondary standards will often be used in a quantitative capacity. Therefore, the
concentration used should be high enough to permit the preparation of dilutions
across the dynamic range of the assay and to allow for dilution into further
matrices. It must be noted that dilutions performed on a high-titre secondary
standard will contribute to the overall uncertainty arising from the dilution
process. This will be the case for most secondary standards.
The target concentration of a standard will be dependent upon its final
intended use and whether any clinical decision points exist when testing for the
analyte. The detectable/quantitative range of all well-established assays for that
pathogen must be taken into consideration. Reference materials that perform
within the dynamic range of an assay (where changes in signal correspond to
changes in analyte concentration) will typically be the norm.
In the case of tertiary standards where the material may be used as an
external run control in qualitative tests, the concentration should ideally be at the
lower end of the range of detection, at a concentration which will appropriately
challenge the assays (for example, three times the 95% limit of detection of a
NAT-based assay, or within the dynamic range of serological assays).
7.5 Volume
The aliquot volume may vary depending on the typical assay input volume for
that analyte and the final storage temperature. The suitability of the container
used for the filling of the aliquots should be validated in terms of the integrity
and stability of the analyte. Where the standard is intended for single use,
as defined in the Instructions for Use provided with the material, sufficient
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volume should be provided for use in the assays for which it is intended and
any remaining material must be discarded by the laboratory as no in-use
stability testing will have been performed. If the standard is for multiple uses,
the volume will depend on the required number of tests, and on stability at the
recommended storage temperature. Where the manufacturer intends a material
to be used on multiple occasions, suitable stability testing should be carried out
to indicate the number of times the material can be removed from its storage
temperature, allowed to warm to ambient temperature and then be re-cooled
before the stability of the material is compromised.
7.7 Infectivity
Use of infectious materials will impact on processing, handling and shipping of
the standard. Where samples are prepared from infectious material, it is important
to provide clear information to the end users about the exact nature of the
material and origin of the pathogens. Import regulations differ from country to
country and can vary depending on the origin of the pathogen. For example,
tissue-culture-derived viral specimens may be subjected to different shipping
regulations by some countries than a patient sample infected with this virus.
WHO Technical Report Series, No. 1004, 2017
7.9 Homogeneity
It is important to confirm the consistency of the filling procedure and to confirm
that the bulk was dispersed (for example, stirred) sufficiently throughout.
Homogeneity is assessed in two ways – by determining both the biological and
the physical content (weight or volume) of multiple vials across the batch. The
latter is particularly important prior to lyophilization, and can be addressed by
weighing a proportion of vials before and after filling and then calculating the
filled weight and associated coefficient of variation as a percentage (% CV). The
% CV will be higher for a more viscous matrix. It is also important to determine
the biological homogeneity by assessing the concentration of the analyte in
multiple vials across the batch. It is known that homogeneity may be impaired
by genetic quasi-species heterogeneity or antibody complexation. The number
of vials used for testing will depend on the batch size. As a minimum, typically
1–2% of the vials should be tested (17).
7.10 Stability
A stability testing programme should be implemented to monitor the potency of
the secondary standard over time. Stability monitoring can be based on real-time
data. However, additional data from accelerated thermal degradation studies
are helpful in characterizing the robustness of lyophilized reference materials.
Such data are also important for assessing the suitability of the material after
extreme shipping conditions.
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lyophilized product may be tested every 6 months for the first year following
production, but further testing could be reduced to annually. Any assessment
of stability and associated outcome should be referenced on the Instructions for
Use distributed with the materials, for example where an acceptable number of
freeze–thaw cycles has been determined, this should be referenced.
by the European Pharmacopoeia (26). The design of the study should take into
consideration that:
■■ each analyte, IS and the candidate secondary standard should be
tested with the same number of dilutions and replicates per dilution;
■■ the adjacent dilution steps should be equally spaced.
The statistical validity of the fitted model should be assessed for each
individual assay. For the parallel-line and Probit models – as the most appropriate
and proven statistical methods for this analysis – the linearity and parallelism
of the logarithmic dose–response relationships between the IS and secondary
standard should be evaluated. If the assay response is linear and the response
lines are parallel then the relative potency of the candidate secondary standard
against the IS can be calculated. Using the parallel-line model, validity criteria
for linearity could include the coefficient of determination (r 2) or a test for
nonlinearity (26). Parallelism could be demonstrated, for example, by means of a
test for non-parallelism or an equivalence approach for the difference or ratio of
slopes (that is, the 95% confidence interval for the ratio of slopes must lie entirely
between predefined equivalence margins). In addition, the precision with which
the potency has been estimated should be provided.
Each calibration will have a stated measurement uncertainty. This
estimate can be determined by identifying all sources of variation, calculating the
extent of variation and using established methods to combine the uncertainty.
The measurement uncertainty associated with assigning a value to the standard
is test-system specific (7, 8). It should be noted that an IS, by definition, has
a specified value which has been assigned and expressed in IU per millilitre
(IU/mL). As a consequence of defining the IU as a fraction of the contents of
the container of the current IS, and because the units defined by any previous IS
formally cease to exist, an uncertainty value is not given to the assigned IU (2).
The variability of the vial weight during filling for each IS is quoted in the study
report and in the Instructions for Use accompanying the standard.
give a number of positive results out of the total number of tests performed.
The Probit analysis will estimate the concentration at which 63% of the samples
tested were positive (that is, the dilution at which on average one single copy per
sample tested could be expected under the assumption of an underlying Poisson
distribution). The calculated end-point is used to give estimates expressed in
NAT-detectable units/mL after correcting for an equivalent volume of the test
sample. It should be noted that these estimates are not necessarily directly
equivalent to a genuine genome equivalent number or copy number per mL.
The software for the Poisson distribution will calculate the proportion of potency
of the test sample (candidate secondary standard) relative to the potency of the
standard sample (that is, the IS) so long as the dose–response curves fit within
the statistical model.
Using a real-time NAT-based assay, the calibration can be determined
from the cycle threshold (Ct) values by applying a parallel-line analysis,
conditional on the assumption that the slope fulfils the requirement of −1/log2.
The number of dilutions and the number of replicates per dilution should follow
the instructions given below in section 8.3.1.
transformation of the assay response may be necessary if the dilution range was
chosen around the sample cut-off rather than the dynamic range of the assay.
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References
1. Guidelines for the preparation and establishment of reference materials and reference reagents
for biological substances. In: WHO Expert Committee on Biological Standardization: twenty-
ninth report. Geneva: World Health Organization; 1978: Annex 4 (WHO Technical Report
Series, No. 626; http://apps.who.int/iris/bitstream/10665/41323/1/WHO_TRS_626.pdf, accessed
5 February 2017).
WHO Technical Report Series, No. 1004, 2017
416
Annex 6
5. International vocabulary of metrology – Basic and general concepts and associated terms
(VIM) third edition. JCGM 200:2012 (http://www.bipm.org/utils/common/documents/jcgm/
JCGM_200_2012.pdf, accessed 5 February 2017).
6. Guidance for industry. Bioanalytical method validation. Rockville (MD): Center for Veterinary
Medicine; 2013 (http://www.fda.gov/downloads/Drugs/Guidance/ucm070107.pdf, accessed
7 February 2017).
7. CLSI. Characterization and qualification of commutable reference materials for laboratory
medicine; approved guideline. CLSI document EP30-A. Wayne (PA): Clinical and Laboratory
Standards Institute; 2010 (sample: http://shop.clsi.org/site/Sample_pdf/EP30A_sample.pdf,
accessed 7 February 2017).
8. ISO 15198:2004. Clinical laboratory medicine – in vitro diagnostic medical devices – validation
of user quality control procedures by the manufacturer. Geneva: ISO; 2004 (abstract: http://www.
iso.org/iso/catalogue_detail.htm?csnumber=39751, accessed 7 February 2017).
9. 2009/886/EC: Commission Decision of 27 November 2009 amending Decision 2002/364/EC
on common technical specifications for in vitro diagnostic medical devices (notified under
document C (2009) 9464) (Text with EEA relevance). European Commission. Official Journal of the
European Union. 2009;L318/25–L318/40 (http://eur-lex.europa.eu/eli/dec/2009/886/oj, accessed
7 February 2017).
10. Frey A, Di Canzio J, Zurakowski D. A statistically defined endpoint titer determination method
for immunoassays. J Immunol Methods. 1998;221(1–2):35–41 (https://www.researchgate.net/
publication/13394025_A_statistically_defined_endpoint_titer_determination_method_for_
Immunoassays, accessed 7 February 2017).
11. ISO 17511:2003. In vitro diagnostic medical devices – measurement of quantities in biological
samples – metrological traceability of values assigned to calibrators and control materials.
Geneva: ISO; 2003 (abstract: http://www.iso.org/iso/iso_catalogue/catalogue_tc/catalogue_detail.
htm?csnumber=30716, accessed 7 February 2017).
12. ISO 18113-1:2009. In vitro diagnostic medical devices – information supplied by the manufacturer
(labelling) – Part 1: Terms, definitions and general requirements. Geneva: ISO; 2009 (https://www.
iso.org/obp/ui/#iso:std:iso:18113:-1:ed-1:v1:en, accessed 7 February 2017).
13. United States Pharmacopeia 38 National Formulary 33 (USP 38-NF 33). United States
Pharmacopeial Convention; 2015.
14. Linden JV, Bianco C, editors. Blood safety and surveillance. New York/Basel: Marcel Dekker, Inc.;
2001.
15. Jung R, Soondrum K, Neumaier M. Quantitative PCR. Clin Chem Lab Med. 2000;38(9):833–6
(abstract: https://www.ncbi.nlm.nih.gov/pubmed/11097336, accessed 7 February 2017).
16. ISO/IEC Guide 98-3:2008. Uncertainty of measurement – Part 3: Guide to the expression of
uncertainty in measurement (GUM:1995). Geneva: ISO; 2009 (abstract: http://www.iso.org/iso/
catalogue_detail.htm?csnumber=50461, accessed 7 February 2017).
17. Resolution WHA63.12. Availability, safety and quality of blood products. Sixty-third World
Health Assembly, Geneva, 17–21 May 2010. Geneva: World Health Organization; 2010 (http://
apps.who.int/gb/ebwha/pdf_files/WHA63-REC1/WHA63_REC1-en.pdf, accessed 7 February 2017).
18. Analytical methods and reference materials program. AMRM Glossary of Terms [website]. NIH, US
Department of Health & Human Services (https://ods.od.nih.gov/Research/AMRMGlossary.aspx,
accessed 7 February 2017).
417
WHO Expert Committee on Biological Standardization Sixty-seventh report
19. ISO 15194:2009. In vitro diagnostic medical devices – measurement of quantities in samples of
biological origin – requirements for certified reference materials and the content of supporting
documentation. Geneva: ISO; 2009 (https://www.iso.org/obp/ui/#iso:std:iso:15194:ed-2:v1:en,
accessed 7 February 2017).
20. Dimech W, Francis B, Kox J, Roberts G. Calculating uncertainty of measurement for serology
assays by use of precision and bias. Clin Chem. 2006;52(3):526–9 (https://www.researchgate.
net/publication/7362486_Calculating_Uncertainty_of_Measurement_for_Serology_Assays_by_
Use_of_Precision_and_Bias, accessed 7 February 2017).
21. Principles and methods of validation of diagnostic assays for infectious diseases. In: Manual of
diagnostic tests and vaccines for terrestrial animals [web version]. Paris: World Organisation for
Animal Health; 2016. Chapter 1.1.6, adopted in May 2013 (http://www.oie.int/fileadmin/Home/
eng/Health_standards/tahm/1.01.06_VALIDATION.pdf), accessed 7 February 2017).
22. Miller WG, Myers GL. Commutability still matters. Clin Chem. 2013;59(9):1291–3 (http://clinchem.
aaccjnls.org/content/59/9/1291, accessed 7 February 2017).
23. WHO Consultation on commutability of WHO Biological Reference Preparations for in vitro
detection of infectious markers. Meeting Report. Geneva: World Health Organization; 2013
(WHO/BS/2013.2230 Addendum 1; http://www.who.int/entity/bloodproducts/norms/BS_2230_
Addendum1_Commutability.pdf?ua=1, accessed 27 March 2017).
24. Kirkwood TB. Predicting the stability of biological standards and products. Biometrics. 1977;
33(4):736–42 (abstract: https://www.ncbi.nlm.nih.gov/pubmed/588659, accessed 7 February
2017).
25. Egan W, Schofield T. Basic principles of stability. Biologicals. 2009;37(6):379–86; and discussion
421–3 (abstract: http://www.sciencedirect.com/science/article/pii/S1045105609001158, accessed
7 February 2017).
26. Statistical analysis of results of biological assays and tests. 01/2008:50300. European
Pharmacopoeia 6.0, section 5.3 (http://www.uspbpep.com/ep60/5.3.%20%20statistical%20
analysis%20of%20results%20of%20biological%20assays%20and%20tests%2050300e.pdf,
accessed 7 February 2017).
27. Konnert A, Berding C, Arends S. Uncertainty calculation for calibrators and controls of laboratory
diagnostic assays. Clin Chem Lab Med. 2006;44(10):1175–82 (abstract: https://www.researchgate.
net/publication/6763360_Uncertainty_calculation_for_calibrators_and_controls_of_laboratory_
diagnostic_assays, accessed 7 February 2017).
WHO Technical Report Series, No. 1004, 2017
28. Bell S. A beginner’s guide to uncertainty of measurement. Measurement good practice guide
No. 11 (Issue 2 with amendments 2001). Teddington: National Physics Laboratory; 1999 (abstract:
http://www.npl.co.uk/publications/a-beginners-guide-to-uncertainty-in-measurement, accessed
7 February 2017).
29. Tang L, Sun Y, Buelow D, Gu Z, Caliendo AM, Pounds S et al. Quantitative assessment of
commutability for clinical viral load testing using a digital PCR-based reference standard.
J Clin Microbiol. 2016;54(6):1616–23 (http://jcm.asm.org/content/54/6/1616.long, accessed
7 February 2017).
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Appendix 1
Example of the parallel calibration of a secondary standard
in a study to establish the International Standard
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WHO/BS/2016.xxxx
ENGLISH ONLY
NOTE:
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should be addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention:
Technologies, Standards and Norms (TSN). Comments may also be submitted electronically to
the Responsible Officer: Dr C M Nübling at email: [email protected].
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be liable for damages arising from its use. The named authors alone are responsible for the views expressed in this publication.
WHO Expert Committee on Biological Standardization Sixty-seventh report
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Summary
This report describes the collaborative study evaluation of the replacement 4th WHO
International Standard for hepatitis B virus (HBV) DNA for the calibration of secondary
reference preparations for HBV nucleic acid amplification techniques (NAT). The candidate 4th
HBV International Standard was prepared and evaluated as part of the collaborative study to
establish the 3rd HBV WHO International Standard in 2011. The lyophilized preparation
comprises a dilution of the same HBV DNA-positive plasma sample as used for the previous
HBV International Standards, in pooled human plasma. In this collaborative study, thirteen
laboratories from seven countries evaluated the suitability of the candidate using their routine
HBV NAT-based assay. The candidate NIBSC code 10/266 (sample 2) was evaluated alongside
the 3rd HBV WHO International Standard, NIBSC code 10/264 (sample 1), three HBV reference
preparations (samples 3-5) and three HBV-positive plasma samples comprising different HBV
genotypes (samples 6-8). A range of HBV NAT assays were used in the evaluation, the majority
of which were commercial quantitative assays based on real-time PCR technology.
The overall mean potency estimates for samples 1 and 2, were 5.94 and 5.97 log10 IU/mL
respectively. These values are very similar to the values obtained for these samples relative to
the pre-existing 2nd HBV WHO International Standard in the 2011 collaborative study (5.93 and
5.98 log10 IU/mL respectively). The standard deviation in individual laboratory mean estimates
for samples 1 and 2 was 0.13 log10 IU/mL. The overall mean potency estimate for sample 2,
relative to sample 1 was 5.96 log10 IU/mL. The results obtained from ongoing accelerated
thermal degradation studies indicate that the candidate sample 2 has remained stable over the 5
years post-manufacture.
The results of the study indicate the suitability of sample 2 as the replacement 4th HBV WHO
International Standard. Since the overall mean potency obtained for the candidate in this
collaborative study is very similar to the overall mean potency obtained in the 2011 collaborative
study, relative to the pre-existing 2nd HBV WHO International Standard, it is proposed that the
value assigned to the candidate sample 2 is that obtained in the 2011 collaborative study. This
approach would minimize any potential drift in the value of the IU during the replacement. It is
therefore proposed that candidate sample 2 (NIBSC code 10/266) is established as the 4th WHO
International Standard for HBV DNA for NAT with an assigned potency of 955,000 IU/mL
(~5.98 log10 IU/mL) when reconstituted in 0.5 mL of nuclease-free water.
Introduction
Hepatitis B virus (HBV) remains a major public health problem worldwide, despite the
availability of an effective vaccine and antiviral therapies. More than 240 million people
worldwide are chronically infected, with 0.5-1 million dying annually as a result of serious liver
disease 1. The virus is transmitted in blood and body fluids, perinatally and through close person-
WHO Technical Report Series, No. 1004, 2017
to-person contact in early childhood (in regions with high HBV prevalence), and through
infected needles and sexual contact (in regions with low HBV prevalence) 1. Nucleic acid
amplification techniques (NAT) for HBV were first introduced for blood screening in 1997, and
are now implemented in at least 30 countries worldwide 2,3. However, there remains a residual
risk of transfusion-transmitted infection, through occult HBV infection and vaccine breakthrough
infections 4. NAT is routinely used in the diagnosis and management of HBV infections,
particularly, to guide the initiation of and monitor the response to antiviral therapy in
chronically-infected patients 5. A range of both commercial and laboratory-developed NAT-
based assays are currently in use. The WHO International Standard for HBV DNA was
established in 1999 6,7, and is used by manufacturers of in vitro diagnostic devices (IVDs), blood
transfusion centres, control authorities, and clinical laboratories, to calibrate secondary reference
materials for NAT in terms of the International Unit (IU).
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The 1st and 2nd WHO International Standards for HBV were prepared by dilution of a Eurohep
R1 sample 8 (Genotype A2, HBsAg subtype adw2, derived from a single donor) in HBV-
negative pooled human plasma. Both materials were prepared from the same bulk (filled and
freeze-dried on two separate occasions) and evaluated in parallel in a worldwide collaborative
study using a range of NAT-based assays for HBV 6,7. The first candidate (NIBSC code 97/746)
was established as the 1st WHO International Standard for HBV DNA in 1999, with an assigned
potency of 1,000,000 IU/mL when reconstituted in 0.5 mL nuclease-free water. In 2006, the
WHO Expert Committee on Biological Standardization (ECBS) established the second candidate
(NIBSC code 97/750) as the replacement 2nd WHO International Standard for HBV DNA
following a smaller collaborative study 9,10. In 2011, two replacement batches (NIBSC codes
10/264 and 10/266) were prepared from the same original HBV Eurohep R1 stock as the 1st and
2nd WHO International Standards, diluted in pooled human plasma, and were evaluated in a
worldwide collaborative study in parallel with the 2nd WHO International Standard for HBV
(NIBSC code 97/750) 11. Mean relative potencies for 10/264 and 10/266 were 5.93 and 5.98
log10 IU/mL respectively, when compared against 97/750. The first candidate (10/264) was
established as the 3rd WHO International Standard for HBV DNA in October 2011 with a unitage
of 850,000 IU/mL. It was noted that the second candidate (10/266) would be a suitable
replacement HBV International Standard in due course, depending on ongoing stability
assessment.
The established use of the HBV IU as the unit of measurement for HBV DNA highlights the
importance of maintaining the availability of this International Standard. This report describes
the collaborative study evaluation of the second candidate 10/266 as the replacement 4th WHO
International Standard for HBV for NAT. The proposal to replace the 3rd WHO International
Standard for HBV DNA was endorsed by the WHO ECBS in October 2015. The collaborative
study results were presented to the Scientific Working Group on the Standardization of Genome
Amplification Techniques (SoGAT) in London in June 2016. The proposed standard is intended
to be used in the in vitro diagnostics field and relates to ISO 17511:2003 Section 5.5 12.
Aims of study
The aim of this collaborative study was to evaluate the suitability of the candidate lyophilized
preparation in parallel with the 3rd HBV WHO International Standard (NIBSC code 10/264)
using a range of NAT-based assays.
Materials
Candidate standard
The candidate preparation (NIBSC code 10/266) comprises lyophilized human plasma and HBV.
The HBV was sourced from a stock of the Eurohep R1 reference material stored at NIBSC and is
a genotype A2, HBsAg subtype adw2 virus from a single donor 8. The pooled human plasma
diluent was sourced from UK blood donations and had been tested and found negative for HIV
antibody, HCV antibody, HBsAg and syphilis. It was also tested at NIBSC and found negative
for HCV RNA by NAT. The preparation was lyophilized to ensure long-term stability.
The filling and lyophilization of the bulk material was performed under contract at eQAD, UK
NEQAS (Colindale, UK), in March 2011 and has been described previously 11. The bulk was
dispensed in 0.5 mL volumes into 3 mL screw-cap glass vials (Adelphi Tubes, Haywards Heath,
UK). The homogeneity of the fill was determined by performing check-weighing of
approximately every fiftieth vial, with vials outside the defined specification being discarded.
Filled vials were partially stoppered, lyophilized and then fully stoppered in the freeze dryer. A
total of 2700 vials were prepared for 10/266. The percentage coefficient of variation (%CV) of
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the fill weight was 0.36%. The sealed vials were returned to NIBSC for storage at -20 °C under
continuous temperature monitoring for the lifetime of the product. Evaluation of multiple
aliquots of 10/266 (n=30) at NIBSC indicated that the HBV content was homogeneous (2SD of
0.12 log10 IU/mL). Comparison of the liquid bulk versus the lyophilized product indicated that
there was a minimal loss in potency of approximately 0.04 log10 IU/mL upon freeze-drying.
Assessments of residual moisture and oxygen content, as an indicator of vial integrity after
sealing, were determined for 20 vials of 10/266 as previously described 11, and were 0.29% (Karl
Fischer, 0.45% NIR units, CV=14.66%) and 0.17% (CV=63.81%) respectively.
Study samples
The lyophilized candidate 10/266 was evaluated alongside the 3rd HBV WHO International
Standard (NIBSC code 10/264), a HBV Secondary Reference Reagent (SRR), a HBV National
Standard (NS) and Working Reagent (WR), and three individual HBV-positive plasma donations
comprising different genotypes. These plasma donations were sourced from HBV-positive
plasma packs rejected by the UK NHS Blood and Tranplant authority (Colindale, UK). The
HBV genotype was determined using a multiplex PCR assay with genotype-specific primers 13.
Lyophilized and liquid frozen study samples were stored at -20 °C and -70 °C, respectively, prior
to shipping to participants by courier on dry ice.
Study design
The aim of this collaborative study was to evaluate the suitability of the candidate 4th HBV
WHO International Standard in parallel with the 3rd WHO International Standard for HBV using
a range of NAT-based assays. Three HBV reference reagents were included in the study with the
intention of calibrating these reagents in IU. Three HBV plasma samples were included in the
study in order to provide a limited assessment of commutability 12,14. Three vials of each study
sample were sent to participating laboratories, with specific instructions for storage,
reconstitution and testing. Samples 6-8 were only sent to laboratories performing quantitative
HBV NAT.
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Study protocol
Participants were requested to test dilutions of each sample using their routine HBV NAT assay
on three separate occasions, using a fresh vial of each sample in each independent assay. In
accordance with the study protocol (Appendix 2), the lyophilized samples were to be
reconstituted with 0.5 mL of deionized, nuclease-free molecular-grade water and left for a
minimum of 20 minutes with occasional agitation before use. Samples 6-8 were to be tested neat
and were therefore only evaluated by laboratories performing quantitative assays.
For quantitative assays, participants were requested to test samples 1-8 neat and to test samples
1-5 at a minimum of two serial ten-fold dilutions (10-1 and 10-2). For qualitative assays,
participants were requested to test ten-fold serial dilutions of samples 1-5, around the assay end-
point (in order to determine the actual assay end-point). For subsequent assays, participants were
asked to test the dilution at the predetermined end-point, and a minimum of two half-log10 serial
dilutions either side of the end-point (i.e., at least five dilutions in total). Participants were
requested to perform dilutions using the sample matrix specific to their individual assay (e.g.
HBV DNA-negative human plasma), and to extract samples prior to HBV DNA measurement.
Participants
Study samples were sent to 13 participants representing 7 countries (Appendix 1). Participants
were selected for their experience in HBV NAT, geographic distribution and participation in
previous evaluation studies. They represented IVD manufacturers, Official Medicines Control
Laboratories (OMCLs) and WHO collaborating centres. All participating laboratories are
referred to by a code number, allocated at random, and not representing the order of listing in
Appendix 1. Where a laboratory returned data using different assay methods, the results were
analyzed separately, as if from different laboratories, and are referred to as, for example,
laboratory 01A, 01B etc.
Statistical methods
Qualitative and quantitative assay results were evaluated separately. In the case of qualitative
assays (from laboratory 12), data from all assays were pooled to give a number positive out of
number tested at each dilution step. A single ‘end-point’ for each dilution series was calculated,
to give an estimate of ‘NAT detectable units/mL’, as described previously 15. It should be noted
that these estimates are not necessarily directly equivalent to a genuine genome copy
number/mL. In the case of quantitative assays, analysis was based on the results supplied by the
participants. Results were reported as IU/mL. For each assay run, a single estimate of log10
IU/mL was obtained for each sample, by taking the mean of the log10 estimates of IU/mL across
replicates, after correcting for any dilution factor. A single estimate for the laboratory and assay
method was then calculated as the mean of the log10 estimates of IU/mL across assay runs.
All analysis was based on the log10 estimates of IU/mL or ‘NAT detectable units/mL’. Overall
mean estimates were calculated as the means of all individual laboratories. Variation between
laboratories (inter-laboratory) was expressed as standard deviation (SD) of the log10 estimates
and % geometric coefficient of variation (%GCV) 16 of the actual estimates. Potencies relative to
sample 1, the current HBV WHO International Standard (10/264), were calculated as the
difference in estimated log10 ‘units per mL’ (test sample – standard) plus the value in log10
IU/mL for the International Standard. Therefore for example, if in an individual assay, the test
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sample is 0.5 log10 higher than the International Standard, assigned 5.93 log10 IU/mL, the relative
potency of the test sample is 6.43 log10 IU/mL.
For the quantitative assays, variation within laboratories, and between assays, (intra-laboratory)
was expressed as SDs and %GCVs of the individual assay mean log10 estimates. These estimates
were pooled across all samples. The significance of the inter-laboratory variation relative to the
intra-laboratory variation was assessed by an analysis of variance.
Samples of 10/266 were stored at elevated temperatures, and assayed at NIBSC in parallel with
samples stored at -20 °C and -70 °C by HBV NAT (as described for the stability of the
lyophilized candidate). Three vials of each sample were evaluated after storage at each
temperature for 11 weeks, 12, 30 and 60 months. It was not possible to reconstitute the vials
stored at +37 °C and +45 °C for 12, 30 and 60 months. The mean estimated log10 IU/mL and
differences (log10 IU/mL) from the -20 °C baseline samples are shown in Table 1. A negative
value indicates a drop in potency relative to the -20 °C baseline. The 95% confidence intervals
for the differences are ±0.08 log10 based on a pooled estimate of the standard deviation between
individual vial test results. As there is no observed drop in potency it is not possible to fit the
usual Arrhenius model for accelerated degradation studies, or obtain any predictions for the
expected loss per year with long-term storage at -20 °C. All available data indicates adequate
stability. Stability testing of 10/266 will be ongoing.
The stability of 10/266 when reconstituted has not been specifically determined. Therefore, it is
recommended that the reconstituted material is for single use only.
Data received
Data were received from all 13 participating laboratories. Participants performed a variety of
different assay methods, with some laboratories performing more than one assay method. In
total, 15 datasets were received from 14 quantitative assays and 1 qualitative assay. Apart from
the cases noted below, there were no exclusions of data.
Quantitative Assays:
Data were returned with dilutions ranging from neat to 10-8 from different laboratories, although
WHO Technical Report Series, No. 1004, 2017
only dilutions between neat and 10-2 were used in the analysis. For Sample 5, one or two
dilutions were removed from the following laboratories’ data, 01, 02B, 03, 05, 06, 07, 08, 10 and
13 because the results were below the limit of detection. Samples not demonstrating dilutional
linearity (i.e. a linear relationship between reported HBV content against log10 dilution with
fitted slope between 0.80 and 1.25) were excluded. Non-linearity was assessed visually and
determined in the following cases; laboratory 11A, Sample 5 on day 2, Samples 3 and 5 on day
3; laboratory 11B, Sample 5 on day 3; laboratory 13, Sample 3 on day 2. Laboratories 04, 05, 07,
09, 11B and 13 had one to three samples with slopes outside the range 0.80-1.25, with the
majority of these cases (7 out of 10) for Sample 5.
Qualitative Assays:
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Laboratory 12 tested multiple dilutions, from 10-1 down to 10-8 for different samples. Only
dilutions that were tested in at least two of the three assays were used in the analysis.
Table 4 shows the overall mean estimates of log10 IU/mL from the quantitative assays, along
with the SD (of log10 estimates) and the %GCV (of actual estimates). The overall mean estimates
for samples 1 and 2 were 5.94 and 5.97 Log10 IU/mL respectively. These values are very similar
to the values obtained for these samples in the 2011 collaborative study 11 (5.93 and 5.98 Log10
IU/mL respectively), despite some differences in the participants and assays involved in each
study. For samples 1-3, the SDs and %GCVs are 0.11-0.13 log10 and 29-35% respectively. The
overall SDs for samples 1 and 2 are slightly higher than those reported in the 2011 collaborative
study. However, this still represents good agreement between laboratories and assay methods.
For samples 4 and 5, the SDs and %GCVs are 0.22-0.27 log10 and 65-86% respectively. For
samples 6-8, the SDs and %GCVs are 0.29-0.42 log10 and 95-160% respectively. The increased
SDs and %GCVs for samples 4-8 are principally due to the outlying results of laboratory 6 (see
Figure 1). The overall mean estimates, SDs and %GCVs for samples 1-8 excluding the dataset
for laboratory 6 are shown in Table 5. The SDs and %GCVs for samples 4-8 are all reduced (by
approximately 2-fold) when the results for laboratory 6 are excluded. Five laboratories reported
results using the cobas® AmpliPrep/cobas® TaqMan® HBV Test, v2.0 (assay code cTM). This
assay is over-represented in comparison to the other assays. However, removing datasets from 3
laboratories using this assay did not greatly alter the overall laboratory results (Table 6)
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Potencies relative to the 3rd WHO International Standard for HBV (Sample 1)
The estimated concentrations of samples 2-8 were expressed in IU, by direct comparison
(relative potencies) to the current International Standard (10/264, sample 1), which has an
assigned unitage of 850,00 IU/mL (5.93 log10), as described in the statistical methods section.
The laboratory mean estimates are shown in Table 7 for the quantitative and qualitative assays.
Units are log10 IU/mL in both cases. The results are also shown in histogram form in Figure 2.
The overall mean relative potencies, along with SDs and %GCVs, are shown in Table 8. The
overall mean relative potency for the candidate sample 2 is 5.96 log10 IU/mL, based on the
quantitative assays. This value compares well to the direct estimate of 5.97 log10 IU/mL from the
quantitative assays which are all calibrated in IU/mL.
Figure 2 and Table 8, show an improvement in the agreement between laboratories for samples
2-5 for the quantitative assays. The SD and %GCV between laboratories has reduced for these
samples when compared to the values in Table 4. The reduction in these values for samples 4
and 5 (both genotype C) is less marked than for samples 1-3 (all genotype A), possibly because
of increased variability in the quantification of genotype C viruses between the assays. For
samples 6-8 there is no improvement in the agreement between laboratories when compared to
the values in Table 4. This may be due to variability in the quantification of different HBV
genotypes, although for sample 8 there is no improvement in the agreement between
laboratories, despite samples 1 and 8 comprising genotype A viruses. Again, the over-
representation of the cTM assay in the study did not greatly alter the overall laboratory results
(Table 9).
Potencies relative to the candidate 4th WHO International Standard for HBV
(Sample 2)
The estimated concentrations of samples 3-8 were expressed in IU, by direct comparison
(relative potencies) to the candidate International Standard (10/266, sample 2), based on a
candidate unitage of 955,000 IU/mL (5.98 log10), as described in the statistical methods section.
This candidate unitage was based on the overall mean potency obtained for 10/266, relative to
the 2nd HBV WHO International Standard (97/750), in the 2011 collaborative study.
Overall mean relative potencies, along with SDs and %GCVs, are shown in Table 10. Table 10
shows an improvement in the agreement between laboratories for samples 3-5 for the
quantitative assays. The SD and %GCV between laboratories has reduced for these samples
when compared to the values in Table 4. For samples 6-8 there is no improvement in the
agreement between laboratories when compared to the values in Table 4. Again, this may be due
to variability in the quantification of different HBV genotypes, although for sample 8 there is no
improvement in the agreement between laboratories, despite samples 2 and 8 comprising
WHO Technical Report Series, No. 1004, 2017
genotype A viruses. The over-representation of the cTM assay in the study did not greatly alter
the overall laboratory results (Table 11).
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the resulting between-assay variability is lower than would be expected if only a single replicate
was tested in each assay. The ‘NAT detectable units’ from the qualitative assays are obtained by
pooling all assay data to give a single series of number positive out of number tested at each
dilution. As a result, there is no comparable analysis of intra-assay variation for the qualitative
assay.
Conclusions
In this study, a range of NAT-based assays for HBV have been used to evaluate the suitability of
the candidate standard (NIBSC code 10/266) as the replacement 4th WHO International Standard
for HBV DNA for NAT-based assays. The candidate was prepared from the same virus stock
used for previous HBV WHO International Standards and was diluted in a similar pooled human
plasma material 6,7,9,10. Production data suggests that the batch is homogeneous and contains
residual moisture and oxygen levels that are within WHO and NIBSC limits for lyophilized
standards 11,17. Comparison of the liquid bulk versus the lyophilized product indicates that there
was minimal loss in potency upon freeze-drying (0.04 log10 IU/mL). The results of ongoing
accelerated thermal degradation studies at 60 months indicate that the candidate is stable and
suitable for long-term use.
In the collaborative study, the lyophilized candidate (sample 2) was evaluated alongside the 3rd
HBV WHO International Standard (sample 1). The overall mean estimates for samples 1 and 2
were 5.94 and 5.97 Log10 IU/mL, respectively, based on the calibration of quantitative assay kits
in IU/mL. These values are very similar to the values obtained for these samples relative to the
2nd HBV WHO International Standard in the 2011 collaborative study 11, despite laboratories
reporting results using different HBV NAT-based assays. In the present study, the agreement
between laboratories for sample 2 was improved when the potency was expressed relative to the
3rd HBV WHO International Standard (sample 1). There was some evidence for variability in the
quantification of different HBV genotypes present in the different study samples. This has been
reported previously 18. Inter-laboratory variability was higher than intra-laboratory variability for
the quantitative assays. This highlights the continued need for standardization of HBV NAT-
based assays, and the importance of accurate calibration to the WHO International Standard.
A full assessment of commutability of the candidate standard for HBV-positive samples has not
been possible in this study, due to the limited number of clinical samples that could be included.
Three HBV-positive plasma samples comprising HBV genotypes A, D and E, from rejected
blood donations were included in the study. There was no overall improvement in the agreement
between laboratories when the estimated concentrations of the three HBV plasma samples were
expressed relative to the candidate 4th HBV WHO International Standard (same formulation as
previous HBV WHO International Standards), compared to the uncorrected values. This is
principally due to variability in the quantification of different HBV genotypes between different
assays, and outlying results from one or two laboratories for samples 6-8. There is no evidence
of non-commutability with the three plasma samples that were included in the study.
In summary, the results of the study indicate the suitability of the candidate sample 2 as the
replacement 4th WHO International Standard for HBV DNA for NAT. Since the overall mean
potency obtained for the candidate in this collaborative study is very similar to the overall mean
potency obtained in the 2011 collaborative study, relative to the pre-existing 2nd HBV WHO
International Standard, it is proposed that the value assigned to the candidate sample 2 is that
obtained in the 2011 collaborative study. This approach would minimize any potential drift in the
value of the IU during the replacement.
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Proposal
It is proposed that the candidate standard, NIBSC code 10/266, is established as the 4th WHO
International Standard for HBV DNA for use in NAT-based assays, with an assigned
potency of 955,000 IU/mL (~5.98 log10 IU/mL) when reconstituted in 0.5 mL of nuclease-free
water. This potency is based on the value that was assigned in the collaborative study in 2011
where the candidate was assessed alongside the 2nd HBV WHO International Standard. The
uncertainty can be derived from the variance of the fill weight and is 0.36%. The proposed
standard is intended to be used by IVD manufacturers, blood transfusion centres, control
authorities, and clinical laboratories, to calibrate secondary reference materials used in HBV
NAT assays. Proposed Instructions for Use (IFU) for the product are included in Appendix 3.
Acknowledgements
We gratefully acknowledge the important contributions of the collaborative study participants.
References
1. World Health Organisation, Hepatitis B, Fact sheet No. 204, updated July 2014.
http://www.who.int/mediacentre/factsheets/fs204/en/
2. Roth WK1, Busch MP, Schuller A, Ismay S, Cheng A, Seed CR, Jungbauer C, Minsk
PM, Sondag-Thull D, Wendel S, Levi JE, Fearon M, Delage G, Xie Y, Jukic I, Turek P,
Ullum H, Tefanova V, Tilk M, Reimal R, Castren J, Naukkarinen M, Assal A, Jork C,
Hourfar MK, Michel P, Offergeld R, Pichl L, Schmidt M, Schottstedt V, Seifried E,
Wagner F, Weber-Schehl M, Politis C, Lin CK, Tsoi WC, O'Riordan J, Gottreich A,
Shinar E, Yahalom V, Velati C, Satake M, Sanad N, Sisene I, Bon AH, Koppelmann M,
Flanagan P, Flesland O, Brojer E, Ltowska M, Nascimento F, Zhiburt E, Chua SS, Teo
D, Stezinar SL, Vermeulen M, Reddy R, Park Q, Castro E, Eiras A, Gonzales Fraile I,
Torres P, Ekermo B, Niederhauser C, Chen H, Oota S, Brant LJ, Eglin R, Jarvis L,
Mohabir L, Brodsky J, Foster G, Jennings C, Notari E, Stramer S, Kessler D, Hillyer C,
Kamel H, Katz L, Taylor C, Panzer S, Reesink HW. International survey on NAT testing
of blood donations: expanding implementation and yield from 1999 to 2009. Vox Sang.
2012;102(1):82-90. doi: 10.1111/j.1423-0410.2011.01506.x.
3. Seo DH, Whang DH, Song EY, Han KS. Occult hepatitis B virus infection and blood
WHO Technical Report Series, No. 1004, 2017
428
Annex 6
WHO/BS/2016.2291
Page 11
7. Saldanha J, Gerlich W, Lelie N, Dawson P, Heermann K, Heath A; WHO Collaborative
Study Group. An international collaborative study to establish a World Health
Organization international standard for hepatitis B virus DNA nucleic acid amplification
techniques. Vox Sang. 2001;80:63-71.
8. Heermann KH, Gerlich WH, Chudy M, Schaefer S, Thomssen R. Quantitative detection
of hepatitis B virus DNA in two international reference plasma preparations. Eurohep
Pathobiology Group. J Clin Microbiol. 1999;37:68-73.
9. Baylis SA, Gerlich W, Chudy M, Pisani G, Klotz A, Kerby S, Heath A. WHO
collaborative study to establish a replacement WHO International Standard for hepatitis
B virus DNA nucleic acid amplification technology (NAT) assays. WHO ECBS Report
2006; WHO/BS/06.2034.
https://extranet.who.int/iris/restricted/bitstream/10665/69957/1/WHO_BS_06.2034_eng.
pdf
10. Baylis SA, Heath AB, Chudy M, Pisani G, Klotz A, Kerby S, Gerlich W. An
international collaborative study to establish the 2nd World Health Organization
International Standard for hepatitis B virus DNA nucleic acid amplification technology-
based assays. Vox Sang. 2008;94:358-62.
11. Fryer JF, Heath AB, Wilkinson DE, Minor PD and the Collaborative Study Group.
Collaborative Study to Evaluate the Proposed 3rd WHO International Standard for
Hepatitis B Virus (HBV) for Nucleic Acid Amplification Technology (NAT)-Based
Assays. WHO ECBS Report 2011;WHO/BS/2011.2170.
http://apps.who.int/iris/bitstream/10665/70779/1/WHO_BS_2011.2170_eng.pdf
12. ISO 17511:2003. In vitro diagnostic medical devices measurement of quantities in
biological samples metrological traceability of values assigned to calibrators and
control materials. Geneva: International Organization for Standardization (ISO); 2003.
13. Kirschberg O, Schüttler C, Repp R, Schaefer S. A multiplex-PCR to identify hepatitis B
virus-genotypes A-F. J Clin Virol. 2004;29:39-43.
14. WHO Consultation on Global Measurement Standards and their use in the in vitro
Biological Diagnostic Field, Switzerland, Geneva, Switzerland, 7-8 June 2004:
http://www.who.int/bloodproducts/publications/en/Minutes-220804.pdf
15. Saldanha J; Lelie N and Heath A B. Establishment of the First International Standard for
Nucleic Acid Amplification Technology (NAT) assays for HCV RNA. 1999. Vox
Sanquinis. 76:149-58.
16. Kirkwood TBL. Geometric means and measures of dispersion. Biometrics 1979;35:908-
9.
17. Recommendations for the preparation, characterization and establishment of international
and other biological reference standards (revised 2004). WHO Technical Report Series
2007. Geneva, Switzerland:WHO 2007; 932,73-131.
http://www.who.int/bloodproducts/publications/TRS932Annex2_Inter_biolefstandardsre
v2004.pdf
18. Chudy M, Hanschmann KM, Kress J, Gerlich W and Nübling CM. Collaborative study to
establish a World Health Organization International Genotype Panel for hepatitis B virus
nucleic acid amplification technique (NAT)-based assays. WHO ECBS Report
2009;WHO/BS/09.2121. http://whqlibdoc.who.int/hq/2009/WHO_BS_09.2121_eng.pdf
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Table 1. Thermal stability of 10/266 at different storage temperatures. * Mean results from
single extractions from 3 vials at each time point and temperature.
Quantitative Assays
Assay Code Assay No. of datasets
kPCR VERSANT HBV DNA 1.0 Assay (kPCR) (Siemens 1
Healthcare Diagnostics)
cTM cobas® AmpliPrep/cobas® TaqMan® HBV Test, 5
v2.0 (Roche Molecular Systems, Inc.)
c68 cobas® HBV test for use on the cobas® 6800/8800 2
Systems (Roche Molecular Systems, Inc.)
AbRT Abbott RealTime HBV (Abbott Molecular, Inc.) 1
ArQS artus® HBV QS-RGQ Kit, Version 1 (QIAGEN) 1
APT Aptima HBV Quant assay on the Panther system 1
(Hologic, Inc.)
VER VERIS HBV Assay (Beckman Coulter, Inc.) 1
SKB HBV DNA real-time PCR detection kit (Shanghai 1
Kehua Bio-Engineering Co., Ltd.)
SaB Hepatitis B Viral DNA Quantitative Fluorescence 1
Diagnostic Kit (PCR Fluorescence Probing) Mag
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Table 3. Laboratory mean estimates from quantitative assays (log10 IU/mL) and qualitative
assays (log10 ‘NAT detectable units/mL’). Qualitative results are shaded in grey. nd, not
determined (either not tested or data excluded).
Table 4. Overall mean estimates and inter-laboratory variation for quantitative assays (log10
IU/mL).
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Table 5. Overall mean estimates and inter-laboratory variation for quantitative assays (log10
IU/mL), excluding the dataset from laboratory 6.
Table 6. Overall mean estimates and inter-laboratory variation for quantitative assays (log10
IU/mL), excluding datasets from laboratories 9, 10 and 13 using the cTM assay.
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Table 7. Laboratory estimates of potency relative to the 3rd WHO International Standard for
HBV (sample 1) from quantitative assays and qualitative assays (log10 IU/mL) - based on an
assigned unitage of the International Standard of 850,000 (5.93 log10) IU/mL. nd, not determined
(either not tested or data excluded).
Table 8. Overall mean estimates and inter-laboratory variation for potency relative to the 3rd
HBV WHO International Standard (sample 1) log10 IU/mL for quantitative assays - based on an
assigned unitage of the International Standard of 850,000 (5.93 log10) IU/mL.
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Table 9. Overall mean estimates and inter-laboratory variation for potency relative to the 3rd
HBV WHO International Standard (sample 1) log10 IU/mL for quantitative assays - based on an
assigned unitage of the International Standard of 850,000 (5.93 log10) IU/mL, excluding datasets
from laboratories 9, 10 and 13 using the cTM assay.
Table 10. Overall mean estimates and inter-laboratory variation for potency relative to the
candidate 4th HBV WHO International Standard (sample 2) log10 IU/mL for quantitative assays -
based on a candidate unitage of 955,000 (5.98 log10) IU/mL.
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Table 11. Overall mean estimates and inter-laboratory variation for potency relative to the
candidate 4th HBV WHO International Standard (sample 2) log10 IU/mL for quantitative assays -
based on a candidate unitage of 955,000 (5.98 log10) IU/mL, excluding datasets from laboratories
9, 10 and 13 using the cTM assay.
Table 12. Intra-laboratory SD of log10 IU/mL and %GCV for quantitative assays.
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Figure legends
Figure 1. Individual laboratory mean estimates for study samples 1-8 (a-h respectively)
obtained using qualitative or quantitative NAT assays. Each box represents the mean estimate
from each laboratory assay and is labeled with the laboratory and assay code. Boxes are also
colour coded by assay.
Figure 2. Relative potencies of samples 2-8 against sample 1 (a-g respectively), for each
qualitative or quantitative assay. Units are expressed as candidate log10 IU/mL. Each box
represents the relative potency for each laboratory assay and is labeled with the laboratory and
assay code. Boxes are also colour coded by assay.
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Figure 1
a Sample 1
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b Sample 2
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c Sample 3
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d Sample 4
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e Sample 5
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f Sample 6
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g Sample 7
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h Sample 8
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Figure 2
a Sample 2 relative to Sample 1
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Appendix 2
Example of the calibration of a national standard
(collaborative study calibration using multiple assays)
Journal of Virological Methods 191 (2013) 122–127
A collaborative study to establish the first National Standard for HIV-1 RNA
nucleic acid amplification techniques (NAT) in Taiwan
Yi-Chen Yang a,b,∗ , Daniel Yang-Chih Shih a , Mong-Hsun Tsai b , Chia-Hung Cheng a , Hwei-Fang Cheng a ,
Chi-Fang Lo a , Der-Yuan Wang a
a
Food and Drug Administration, Department of Health, Executive Yuan, Taipei 11561, Taiwan
b
Institute of Biotechnology, National Taiwan University, Taipei 10672, Taiwan
a b s t r a c t
Article history: The World Health Organization (WHO) International Standard (IS) for human immunodeficiency virus
Received 5 January 2013 type 1 (HIV-1) RNA is only available in limited amounts. It is critical to use the most common HIV-1
Received in revised form 26 March 2013 genotype as source for secondary standards, e.g. a National Standard for Taiwan. The objective of this
Accepted 4 April 2013
study was to establish the first National Standard for HIV-1 RNA NAT assays in Taiwan. A collaborative
Available online 19 April 2013
study, including eleven laboratories from five different countries, was carried out to establish the HIV-1
RNA National Standard by calibration, in International Units (IU), against the WHO HIV-1 RNA IS. The
Keywords:
HIV-1 RNA content for the candidate was quantitated by each laboratory in three independent assays
Standard
Nucleic acid amplification techniques (NAT)
and the results were collected and analyzed statistically. Overall, a high level of agreement among results
Human immunodeficiency virus type 1 was achieved from different laboratories. In addition, the stability study indicated that the candidate was
(HIV-1) stable for 24 months at −80 ± 5 ◦ C. In conclusion, the candidate standard was established as the first
National Standard for HIV-1 RNA for use in NAT assays in Taiwan. The standard is intended to be used for
the quality control of HIV-1 NAT assays and as a quantitative reference material for HIV-1 NAT assays.
© 2013 Elsevier B.V. All rights reserved.
WHO Technical Report Series, No. 1004, 2017
Y.-C. Yang et al. / Journal of Virological Methods 191 (2013) 122–127 123
based on NAT technology have been developed to detect HIV proposed national candidate standard and WHO IS for HIV-1 RNA
qualitatively or quantitatively for blood screening or viral load mea- (97/650) and were requested to perform three independent assays
surement. Such NAT-based IVDs are highly related to blood safety, for HIV-1 RNA using the candidate standard and the WHO IS. For
the quality and performance of the IVDs are of great importance. In each assay, serial dilutions of the WHO IS were prepared using the
order to ensure the continued fitness for purpose of the IVD, both appropriate diluent. The recommended diluent for the study was
the pre-market approval testing and the performance evaluation HIV-1 RNA negative human plasma. The serially diluted IS were
are crucial in post-marketing surveillances. used to calibrate the candidate standard (Sample A) by creating a
The first and second World Health Organization (WHO) Inter- standard curve. If a commercial kit was used, the IS could be treated
national Standard (IS) for HIV-1 RNA was established by the WHO as a second unknown sample (sample B) and quantitated in parallel
Expert Committee on Biological Standardization (ECBS) in 1999 with sample A. A single estimate was obtained for each sample in
and 2008, and the National Institute for Biological Standards and each laboratory and for each assay method by calculating the geo-
Control (NIBSC) code numbers are 97/656 and 97/650, respectively metric mean of repeat data within a single assay. The overall mean
(Holmes et al., 2001; Davis et al., 2008). One of the main purposes and SD were then calculated from the results of all participants.
of an International Standard is to facilitate the calibration of sec-
ondary working reagents developed at a local level, i.e. by individual 2.3. Stability study on the candidate standard
laboratories. The calibration of secondary working reagents from
a higher order standard would help reduce test result variability Vials of the proposed national candidate standard were incu-
from different laboratories, aid in comparing the different com- bated at +4 ◦ C, +24 ◦ C, −20 ◦ C, and −80 ◦ C, three vials were removed
mercial and ‘in-house’ assays, and make it easier to compare the at regular intervals for three independent tests. Two different com-
proficiency of different laboratories (Revets et al., 1996; Schuurman mercial assays were used for quantitative analysis: the Abbott
et al., 1996). In order to ensure the correct use of the Interna- RealTime HIV-1 (Abbott Molecular Inc., Des Plaines, IL, USA) and
tional Standard by the end user, for example for secondary working the COBAS Ampliprep/COBAS TaqMan HIV-1 Test, v1.0 (Roche
reagent calibration and not for use as a routine run control, the Molecular Systems, Inc., Branchburg, NJ, USA). Here, 500 uL of the
WHO IS for HIV-1 RNA is available only in limited amounts, several HIV-1 RNA candidate standard was used in Abbott RealTime HIV-
control laboratories (such as National Institute for Biological Stan- 1 Kit and 1 mL 5× pre-diluted candidate standard was used in
dards and Control (NIBSC), Food and Drug Administration (FDA) COBAS Ampliprep/COBAS TaqMan HIV-1 Test Kit. Both real-time
and Istituto Superiore di Sanità (ISS)) have already prepared an PCR systems, the Abbott m2000 RealTime system and the COBAS
in house or national secondary HIV-1 NAT working reagent them- Ampliprep/TaqMan 48 system, were used according to the manu-
selves (Davis et al., 2003; Lee et al., 2006; Pisani et al., 2007). It facturer’s instructions.
is known that the distribution of HIV subtypes may differ by geo-
graphic region. It is therefore critical to use the major genotype of 3. Results
the HIV-1 as a source material for a National Standard. Since sub-
type B was found to be the predominant genotype in Taiwan, the 3.1. Assay methods
genotype of the National Standard would select to be subtype B, the
same as WHO IS. Therefore, the objective of this study was to estab- Ten of the participants performed quantitative assays: four
lish the first National Standard for HIV-1 RNA NAT assays and to laboratories used the Abbott RT HIV-1 (Abbott Molecular
calibrate the HIV-1 RNA content of the candidate standard against Inc., Des Plaines, IL, USA); two laboratories used the COBAS
the WHO IS for HIV-1 RNA NAT assays (97/650). The procedure for AmpliPrep/COBAS TaqMan HIV-1 Test, v1.0 (Roche Molecular Sys-
the development of a National Standard was based on the previous tems, Inc., Branchburg, NJ, USA); two laboratories used the COBAS
experience of the development of the National Standard for human AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 (Roche Molecular Sys-
parvovirus B19 DNA nucleic acid amplification techniques (NAT) in tems, Inc., Branchburg, NJ, USA); two laboratories used the COBAS
2008 (Yang et al., 2008). TaqMan HIV-1 Test, for use with High Pure System Viral Nucleic
Acid Kit, v1.0 (Roche Molecular Systems, Inc., Branchburg, NJ,
USA); two laboratories used the VERSANT HIV-1 RNA 3.0 (Siemens
2. Materials and methods
Healthcare Diagnostics Inc., Tarrytown, NY, USA); and one labo-
ratory used the COBAS Amplicor HIV-1 Monitor Test, v1.5 (Roche
2.1. Preparation of the candidate standard
Molecular Systems, Inc., Branchburg, NJ, USA). Between them, lab
code 1 used two different methods to detect HIV-1RNA and ana-
The candidate standard for HIV-1 RNA NAT assays was liquid
lyzed the data separately (1A, 1B), and lab code 2 used three
preparation and stored at or below −70 ◦ C. It was prepared by dilut-
different methods to detect HIV-1RNA and reported the results
ing HIV-1 RNA positive plasma in pooled human cryosupernatant.
separately (2A, 2B, 2C). The overall results were therefore based
The proposed titer was approximately 104 IU/mL. The original HIV-
on a maximum of 13 data sets. All these data sets were obtained
1 RNA positive plasma had a titer of HIV-1 RNA of approximately
by commercial assays. The quantitative methods used are summa-
4.7 × 104 IU/mL and was negative for HBsAg, HBV DNA, anti-HCV,
rized in Table 1. The other one participant used the COBAS HIV-1
HCV RNA, HAV RNA as well as B19V DNA. The genotype of the HIV-
AmpliScreen Test, v1.5 (Roche Molecular Systems, Inc., Branchburg,
1 RNA positive plasma was confirmed as subtype B by sequencing.
NJ, USA), which is a qualitative assay that was only give “positive”
The cryosupernatant was negative for HBsAg, HBV DNA, B19V DNA,
results (Detection limit: 78.4 IU/mL) and could not be calculated.
anti-HCV, HCV RNA, anti-HIV 1/2, HIV-1 RNA, and HAV RNA.
3.2. Estimated value of the HIV-1 RNA for the candidate standard
2.2. Design of the international collaborative study
The estimated values of HIV-1 RNA, relative to the International
The aim of the international collaborative study was to calibrate Standard, for the candidate standards from each laboratory are
the titers of the HIV-1 RNA National Standard that was prepared listed in Table 2 and shown in Fig. 1. All the values have shown a
by the Taiwan Food and Drug Administration (TFDA). Including good agreement with each other, except one laboratory has submit-
our laboratory, a total of eleven laboratories from five different ted an outlying result. The value of HIV-1 RNA estimate from each
countries have participated in this study. Participants received the laboratory is shown in Fig. 2. Each box represents the estimate from
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124 Y.-C. Yang et al. / Journal of Virological Methods 191 (2013) 122–127
Table 1
An overview of the quantitative assays used in the collaborative study.
2B COBAS TaqMan HIV-1 Test, for use with A highly conserved region in
10 High Pure System Viral Nucleic Acid Kit, v1.0 HIV-1 gag p41 gene
9 COBAS Amplicor HIV-1 Monitor Test, v1.5 A highly conserved regions in HIV-1 gag gene
A Table 2
8 The estimated values of HIV-1 RNA (Log IU/mL)a for candidate standard from 10
7 Mean(4.04) laboratories.
-2SD(3.66)
6 +2SD(4.42)
Lab code Mean Minimum Maximum CV (%)
Log IU/mL
5
4 1A 4.04 4.03 4.06 0.37
3 1B 3.98 3.96 4.01 0.58
2A 4.13 4.06 4.23 1.37
2
2B 4.11 4.01 4.18 1.63
1
2C 4.13 4.06 4.24 1.79
0 3 4.15 4.12 4.17 0.63
1A 1B 2A 2B 2C 3 4 5 6 7 8 9 10
4 3.73 3.67 3.79 1.04
Lab code 5 4.03 3.99 4.08 1.09
6 3.98 3.84 4.08 4.26
B 7 4.18 4.04 4.26 2.83
8 8 3.95 3.90 4.03 1.84
Mean(4.01)
7 9 – 4.42b 4.42b –
-2SD(3.69)
6 +2SD(4.33) 10 3.68 3.65 3.70 0.59
Log IU/mL
5 a
The measurements were performed using WHO International Standard for
4 human HIV-1 RNA (WHO IS, 97/650) as the standard.
b
3 Only one assay result was available from this laboratory, not three independent
2 assay results.
1
0
1A 1B 2A 2B 2C 3 4 5 6 7 8 9 10
one laboratory and/or assay method. All data were within a range of
Lab code 1.0 Log for each sample, indicating that all the laboratories were in
good agreement with the estimates. A comparison of the different
Fig. 1. The estimated values of HIV-1 RNA (Log IU/mL) for candidate standard as
determined by 10 laboratories (A) and by 9 laboratories (B). The results showed commercial kit results is shown in Table 3; the results showed that
that all the laboratories were in good agreement with the estimates, except one the COBAS Amplicor HIV-1 Monitor Test, v1.5 was significantly dif-
laboratory submitted an outlying result (A). The data generated from Lab code 9 ferent from other commercial assay kits. The data generated from
was excluded from the overall means for the candidate standard (B). the COBAS Amplicor HIV-1 Monitor Test was therefore excluded
from the overall means for the candidate standard. Therefore, the
WHO Technical Report Series, No. 1004, 2017
Fig. 2. The histogram of estimated values of HIV-1 RNA (Log10 IU/mL) for candidate standard from 10 laboratories. The number labeled in the box represented the laboratory
code number.
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Y.-C. Yang et al. / Journal of Virological Methods 191 (2013) 122–127 125
A 4. Discussion
candidate 24°C
8 There was a clear variation between the results from earlier HIV-
candidate original value
7
6 1 viral load assays such as nucleic acid signal branch amplification
Log IU/mL
5 (NASBA), PCR end point detection and branched DNA (bDNA) sig-
4 nal amplification compared to more recent tests such as the Abbott
3
2 real-time assay. The limitation of these assays has previously been
1 reported (Church et al., 2011) and it is known that are optimized to
0 target subtype B group M viruses. New-generation real-time PCR
0 1 2 3 4 5 6 7 8 9
assays for HIV-1 RNA quantification include the Abbott RT HIV-1
Time (weeks)
assay and the Cobas Ampli-Prep/Cobas TaqMan HIV-1 assay (CAP-
B
CTM). These real-time PCR assays have been improved and are
candidate 4°C
8 candidate original value
now able to detect HIV-1 group M, non-B subtype viruses, group
7 N viruses and O viruses. In addition, the Abbott RealTime HIV-1
Log IU/mL
6
5 assay has been reported to successfully detect HIV-1 group P infec-
4 tion (Plantier et al., 2009). However, it has also been reported that
3
2 multiple mismatches in gag primers and probe binding regions for
1 the first version of the CTM assay (CTM1) exist, which can result in
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 an underestimation of the CTM1 values for some patients infected
Time (weeks) with HIV-1 group M, non-B subtypes. To overcome this problem,
Roche Diagnostics have already upgraded their test to CTM version
C
2.0 (CTM2), which uses a dual-target strategy (Damond et al., 2010;
candidate -20°C
Church et al., 2011; Wirden et al., 2011).
8 candidate original value
7 In this international collaborative study, most of the partici-
Log IU/mL
6
5 Test, v1.5 was significantly different from other commercial assay
4 kits. The data generated from the COBAS Amplicor HIV-1 Monitor
3
2 Test was therefore excluded from the overall means for the candi-
1
0 date sample. Since there is only one assay result available in this
0 2 4 6 8 10 12 14 16 18 20 22 24 collaborative study, it does not represent the performance of the
Time (months) kit. Interestingly, a similar result was also shown in an earlier col-
laborative study to establish a replacement International Standard
Fig. 3. Stability analysis of HIV-1 RNA present in candidate standard after storage at
for HIV-1 RNA nucleic acid assays (Davis et al., 2008).
different temperatures: (A) +24 ◦ C for 8 weeks, (B) +4 ◦ C for 12 weeks, (C) −20 ◦ C for
24 months and (D) −80 ◦ C for 24 months. The solid line and the dotted line represent All data points received from laboratories were within a range
the tested values and the original values before storage, respectively. of 1.0 Log from this collaborative study, furthermore, most of the
data were within in a range from 3.9 Log IU/mL to 4.1 Log IU/mL. In
conclusion, a high level of agreement among the results obtained
overall mean for the candidate standard is 1.0 × 104 IU/mL, and the from the participating laboratories was observed. The first National
95% confidence intervals is 8.26 × 103 to 1.25 × 104 IU/mL (Table 4). Taiwan Standard for HIV-1 RNA NAT assays, with an assigned value
of 1.0 × 104 IU/mL, was recognized. In order to reflect the pre-
3.3. Stability analysis of the candidate standard dominant HIV-1 subtype found in Taiwan, this National Standard
was formulated from a subtype B plasma. The results of the sta-
Several vials of the candidate standard were stored at +24 ◦ C, bility study indicated that the HIV-1 RNA National Standard is
+4 ◦ C, −20 ◦ C, and −80 ◦ C, three vials were randomly selected for stable long-term when stored at −20 ◦ C and -80 ◦ C. Therefore, the
stability tests. Samples were taken after one-, two- or four-week first National Standard for HIV-1 RNA NAT assays in Taiwan was
intervals from candidate samples stored at +24 ◦ C and +4 ◦ C and established. This standard could be used for quality control of HIV-
after three- or six-month intervals from candidate samples stored 1 RNA assays and as a quantitative reference material for HIV-1
at −20 ◦ C and −80 ◦ C. Triplicate samples were assayed for each NAT assays. Moreover, the standard could be used nationally for
time point at different temperatures in three independent tests. pre-market approval testing and the performance evaluation in
The calculated mean concentration (IU/mL) for each time point post-marketing surveillances of NAT-based IVDs and facilitating to
and temperature is shown in Fig. 3. The results indicate that the ensure the continued fitness for purpose of the IVD, either imported
candidate samples were stable after storage at +24 ◦ C for 4 weeks, or domestic.
at +4 ◦ C for 8 weeks, at −20 ◦ C for 24 months, and at −80 ◦ C for In recent years, subtype CRF 07 BC has been the major group
24 months. The results suggest good long term stability for the of HIV-1 found in the intravenous drug abuser population in
proposed national candidate standard when stored at −20 ◦ C and Taiwan. As HIV-1 strain diversity and viral recombination events
−80 ◦ C. increase, the need for surveillance using commercial assays to
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126 Y.-C. Yang et al. / Journal of Virological Methods 191 (2013) 122–127
Table 3
Comparison of different commercial kits.
COBAS TaqMan HIV-1 test, for use with High 2B 4.11 3.90 0.30 7.81
Pure System Viral Nucleic Acid Kit, v1.0 10 3.68
a a
COBAS Amplicor HIV-1 Monitor Test, v1.5 9 4.42 4.42
Table 4
Overall mean estimates of HIV-1 RNA (Log IU/mL) for candidate standard.
ensure detection ability and viral load monitoring accuracy in HIV- - Lena Panagiotopoulos/Stirling Dick, National Serology Reference
1-infected patients has increased. To fulfill this goal, an HIV-1 CRF Laboratory (NRL), Australia.
07 BC National standard will need to be established as the next - Micha Nübling/Michael Chudy, Paul-Ehrlich-Institute, Germany.
major step. - Yi-Li Shih, E-Da Hospital, Taiwan.
Disclaimer References
The findings and conclusions in this article have not been for- Bolinger, C., Boris-Lawrie, K., 2009. Mechanisms employed by retroviruses to exploit
mally disseminated by Taiwan FDA and should not be construed to host factors for translational control of a complicated proteome. Retrovirology
6, 8.
represent any agency determination or policy. Busch, M.P., Dodd, R.Y., 2000. NAT and blood safety: what is the paradigm? Trans-
fusion 40, 1157–1160.
Church, D., Gregson, D., Lloyd, T., Klein, M., Beckthold, B., Laupland, K., Gill, M.J., 2011.
Acknowledgements Comparison of the RealTime HIV-1, COBAS TaqMan 48 v1.0, Easy Q v1.2, and
Versant v3.0 assays for determination of HIV-1 viral loads in a cohort of Canadian
patients with diverse HIV subtype infections. J. Clin. Microbiol. 49, 118–124.
We would like to express our sincere thanks to all the par- Damond, F., Avettand-Fenoel, V., Collin, G., Roquebert, B., Plantier, J.C., Ganon, A.,
ticipants of the collaborative study group (Appendix A) for their Sizmann, D., Babiel, R., Glaubitz, J., Chaix, M.L., Brun-Vezinet, F., Descamps,
valuable contribution to this collaborative study and to Ai-Zhen D., Rouzioux, C., 2010. Evaluation of an upgraded version of the Roche Cobas
AmpliPrep/Cobas TaqMan HIV-1 test for HIV-1 load quantification. J. Clin. Micro-
Yang and Yu-Chen Pao for their technical assistance for formulat-
biol. 48, 1413–1416.
ing the candidate standard. We would like to thank Clare Morris Davis, C., Heath, A., Best, S., Hewlett, I., Lelie, N., Schuurman, R., Holmes, H., 2003.
for providing language help. In addition, we also acknowledge the Calibration of HIV-1 working reagents for nucleic acid amplification techniques
funding support from Taiwan FDA. against the 1st international standard for HIV-1 RNA. J. Virol. Methods 107,
37–44.
Davis, C., Berry, N., Heath, A., Holmes, H., 2008. An international collaborative study
to establish a replacement World Health Organization International Standard
Appendix A. for human immunodeficiency virus 1 RNA nucleic acid assays. Vox Sang. 95,
218–225.
WHO Technical Report Series, No. 1004, 2017
List of participants: Holmes, H., Davis, C., Heath, A., Hewlett, I., Lelie, N., 2001. An international collabo-
rative study to establish the 1st international standard for HIV-1 RNA for use in
nucleic acid-based techniques. J. Virol. Methods 92, 141–150.
- Brian Erickson, Abbott Molecular Inc., USA. Karlsson Hedestam, G.B., Fouchier, R.A., Phogat, S., Burton, D.R., Sodroski, J., Wyatt,
R.T., 2008. The challenges of eliciting neutralizing antibodies to HIV-1 and to
- Chien-Feng Sun/Shuan Yang, Chang-Gung Memorial Hospital, influenza virus. Nat. Rev. Microbiol. 6, 143–155.
Taiwan. Lee, S., Wood, O., Taffs, R.E., Hu, J., Machuca, A., Vallejo, A., Hewlett, I., 2006. Devel-
- Clare Morris, National Institute for Biological Standards and Con- opment and evaluation of HIV-1 subtype RNA panels for the standardization of
HIV-1 NAT assays. J. Virol. Methods 137, 287–291.
trols (NIBSC), UK.
Lin, Y.T., Lan, Y.C., Chen, Y.J., Huang, Y.H., Lee, C.M., Liu, T.T., Wong, W.W., Yang,
- Chih-Yuan Yang/Cheng-Feng Kao, Centers for Disease Control J.Y., Wang, C.T., Chen, Y.M., 2007. Molecular epidemiology of HIV-1 infection
(TCDC), Taiwan. and full-length genomic analysis of circulating recombinant form 07 BC strains
- Der-Yuan Wang/Yi-Chen Yang, Food and Drug Administration from injection drug users in Taiwan. J. Infect. Dis. 195, 1283–1293.
Murthy, K.K., Henrard, D.R., Eichberg, J.W., Cobb, K.E., Busch, M.P., Allain, J.P.,
(TFDA), Taiwan. Alter, H.J., 1999. Redefining the HIV-infectious window period in the chim-
- Eric T. Natoli, Siemens Healthcare Diagnostics, USA. panzee model: evidence to suggest that viral nucleic acid testing can prevent
- Indira Hewlett/Sherwin Lee, Center for Biologics Evaluation and blood–borne transmission. Transfusion 39, 688–693.
Piatak Jr., M., Saag, M.S., Yang, L.C., Clark, S.J., Kappes, J.C., Luk, K.C., Hahn, B.H.,
Research/Food and Drug Administration (CBER/FDA), USA. Shaw, G.M., Lifson, J.D., 1993. High levels of HIV-1 in plasma during all stages
- John Saldanha/Matthew Lin, Roche Molecular Systems, USA. of infection determined by competitive PCR. Science 259, 1749–1754.
448
Annex 6
Y.-C. Yang et al. / Journal of Virological Methods 191 (2013) 122–127 127
Pisani, G., Marino, F., Cristiano, K., Bisso, G.M., Mele, C., Luciani, F., Wirz, M., Gentili, Simon, F., Mauclere, P., Roques, P., Loussert-Ajaka, I., Muller-Trutwin, M.C., Saragosti,
G., 2007. Collaborative study for the calibration of HCV RNA, HBV DNA and HIV S., Georges-Courbot, M.C., Barre-Sinoussi, F., Brun-Vezinet, F., 1998. Identifica-
RNA reference preparations against the relative international standards. Ann. tion of a new human immunodeficiency virus type 1 distinct from group M and
Ist. Super. Sanita 43, 69–76. group O. Nat. Med. 4, 1032–1037.
Plantier, J.C., Leoz, M., Dickerson, J.E., De Oliveira, F., Cordonnier, F., Lemee, V., Spira, S., Wainberg, M.A., Loemba, H., Turner, D., Brenner, B.G., 2003. Impact of clade
Damond, F., Robertson, D.L., Simon, F., 2009. A new human immunodeficiency diversity on HIV-1 virulence, antiretroviral drug sensitivity and drug resistance.
virus derived from gorillas. Nat. Med. 15, 871–872. J. Antimicrob. Chemother. 51, 229–240.
Pluta, K., Kacprzak, M.M., 2009. Use of HIV as a gene transfer vector. Acta Biochim. Statistics of HIV/AIDS 12/31/2012. Center for Disease Control, ROC (Taiwan). 2012,
Pol. 56, 531–595. 1.
Revets, H., Marissens, D., de Wit, S., Lacor, P., Clumeck, N., Lauwers, S., Zissis, G., UNAIDS report on the global AIDS epidemic 2012. Joint United Nations Programme
1996. Comparative evaluation of NASBA HIV-1 RNA QT, AMPLICOR-HIV monitor, on HIV/AIDS, pp. 8–14.
and QUANTIPLEX HIV RNA assay, three methods for quantification of human Weiss, R.A., 1993. How does HIV cause AIDS? Science 260, 1273–1279.
immunodeficiency virus type 1 RNA in plasma. J. Clin. Microbiol. 34, 1058–1064. Wirden, M., Tubiana, R., Fourati, S., Thevenin, M., Simon, A., Canestri, A., Ait-
Robertson, D.L., Anderson, J.P., Bradac, J.A., Carr, J.K., Foley, B., Funkhouser, R.K., Gao, Arkoub, Z., Soulie, C., Marcelin, A.G., Katlama, C., Calvez, V., 2011. Upgraded
F., Hahn, B.H., Kalish, M.L., Kuiken, C., Learn, G.H., Leitner, T., McCutchan, F., Cobas Ampliprep-Cobas TaqMan version 2.0 HIV-1 RNA quantification assay
Osmanov, S., Peeters, M., Pieniazek, D., Salminen, M., Sharp, P.M., Wolinsky, S., versus first version: correction of underestimations. J. Clin. Microbiol. 49,
Korber, B., 2000. HIV-1 nomenclature proposal. Science 288, 55–56. 2700–2702.
Schuurman, R., Descamps, D., Weverling, G.J., Kaye, S., Tijnagel, J., Williams, I., Yang, Y.C., Chang, C.J., Tseng, C.I., Wang, D.Y., Cheng, H.F., Lin, C.P., 2008. Establish-
van Leeuwen, R., Tedder, R., Boucher, C.A., Brun-Vezinet, F., Loveday, C., 1996. ment of a National Taiwan Standard and working reagent for human parvovirus
Multicenter comparison of three commercial methods for quantification of B19 DNA nucleic acid amplification techniques (NAT). J. Food Drug Anal. 16,
human immunodeficiency virus type 1 RNA in plasma. J. Clin. Microbiol. 34, 9–14.
3016–3022.
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Appendix 3
Example of the calibration of a reference preparation by a
single NAT assay
The reference preparation (RP) is calibrated against the WHO IS by testing
dilutions of both samples using a real-time PCR, collecting raw data (threshold
cycle values Ct) and performing a valid statistical analysis (e.g. parallel line).
Study performed under the supervision of G. Pisani, ISS, Rome, Italy.
Study samples:
■■ RP for HIV RNA with a presumptive titre of 15 000 IU/mL of
HIV RNA
■■ 3rd WHO IS HIV RNA batch 10/152 with a concentration of
185 000 IU/mL (5.26 log IU/mL).
Test the following dilutions of WHO IS and RP (in triplicate) on three separate
days. Collect the raw data.
Day 1
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Day 2
Day 3
Sample Final dilution Concentration Raw data (Ct value)
(log)
Replica 1 Replica 2 Replica 3
WHO IS −1.09 15.000 IU/mL 28.6 28.3 28.2
−1.59 4.700 IU/mL 29.1 29.3 30.1
−2.09 1.500 IU/mL 31.1 31.2 31.5
−2.59 470 IU/mL 33.4 33.3 32.5
RP Not diluted – 28.8 28.9 28.7
−0.50 – 30.1 30.5 30.2
−1.00 – 32.1 32.2 31.9
−1.50 – 33.5 33.2 34.3
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Annex 7
Guidelines on regulatory preparedness for provision of
marketing authorization of human pandemic influenza
vaccines in non-vaccine-producing countries
1. Introduction 460
2. Purpose and scope 461
3. Terminology 462
4. General considerations for regulatory preparedness for pandemic
influenza vaccines 463
4.1 Acknowledgement of the role of the NRA in the national pandemic influenza
preparedness plan 464
4.2 Considerations for national regulatory preparedness 464
4.3 Reliance on the decisions and expertise of other regulatory authorities 466
4.4 Seasonal influenza vaccines and pandemic preparedness influenza vaccines 467
5. Regulatory evaluation processes 468
5.1 Expected basic documentation according to the source of pandemic
influenza vaccine 470
5.2 Possible regulatory review processes in a pandemic emergency 471
5.3 WHO collaborative procedure for prequalified vaccines 474
5.4 Final evaluation 475
5.5 Emergency approval 476
5.6 Post-marketing risk management and surveillance 476
6. Quality control preparedness 477
Authors and acknowledgements 477
References 479
Appendix 1 Checklist of regulatory actions for pandemic influenza preparedness
and response 482
Appendix 2 Examples of information and documentation that may be required for
the evaluation of a seasonal influenza vaccine annual virus strain change 484
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458
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Abbreviations
CTD Common Technical Document
GMP good manufacturing practice(s)
NCL national control laboratory
NRA national regulatory authority
PIP Pandemic Influenza Preparedness (Framework)
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1. Introduction
An influenza pandemic occurs when a novel influenza A virus emerges against
which most people do not have immunity, and spreads rapidly around the world.
A pandemic influenza A virus is significantly different from normally circulating
human influenza A viruses, with a widespread absence of immunity against the
virus observed in the population. As with seasonal influenza viruses, pandemic
influenza viruses have the ability to spread easily from human to human and
cause disease. This may result in several simultaneous epidemics worldwide with
high numbers of cases of clinical disease and deaths, leading to considerable
social disruption. Pandemic influenza viruses may evolve from subtypes that
previously only circulated in animals or from subtypes currently circulating
in humans but sufficiently different antigenically for pre-existing immunity in
the population to be low or minimal (an example of the latter case is the 2009
H1N1 influenza pandemic). Influenza viruses that have caused past pandemics
have typically originated from animals. Owing to the urgent public health need,
strategies to shorten the time between the emergence of a human pandemic
influenza virus and the availability of safe and effective pandemic influenza
vaccines are one of the highest priorities in global health security.
The WHO Guidelines on regulatory preparedness for human pandemic
influenza vaccines (1) were adopted by the WHO Expert Committee on
Biological Standardization in 2007. These Guidelines provide national regulatory
authorities (NRAs) and vaccine manufacturers with:
■■ guidance regarding regulatory pathways for approving pandemic
influenza vaccines;
■■ the regulatory considerations to take into account when evaluating
the quality, safety and efficacy of candidate vaccines;
■■ guidance on effective post-marketing surveillance of pandemic
WHO Technical Report Series, No. 1004, 2017
influenza vaccines.
The Guidelines apply mainly to countries where influenza vaccine
production takes place, but also contain much information that can be useful
for countries in which vaccines are not produced (hereafter referred to as
non-vaccine-producing countries). However, consultations with stakeholders
following the 2009 H1N1 influenza pandemic identified lack of regulatory
preparedness as one of the factors that delayed or prevented the deployment
of pandemic influenza vaccine in non-vaccine-producing countries. This was
especially the case for vaccine destined for donation or deployed by United
Nations agencies in response to the pandemic emergency (2–4).
The present Guidelines were developed in response to requests
from non-vaccine-producing countries for guidance on the identification of
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3. Terminology
The definitions given below apply to the terms as used in these WHO Guidelines.
These terms may have different meanings in other contexts.
Alert phase: the phase during which influenza caused by a new strain
is identified in humans. Increased vigilance and careful risk assessment at local,
national and global levels are characteristic of this phase (6).
Influenza pandemic: an influenza pandemic (or global epidemic) occurs
when a novel influenza virus strain appears which is significantly different from
circulating strains and against which almost no one is immune. The Director-
General of WHO may, as appropriate, declare a public health emergency of
international concern under the International Health Regulations (2005) following
the identification and determination of global spread of human influenza caused
by a new virus strain (6, 7).
Interpandemic phase: the period between influenza pandemics (6).
Marketing authorization: a formal authorization for a medicine to be
marketed. Once an NRA approves a marketing authorization application for a
new medicine, the medicine may be marketed and made available for physicians
to prescribe. Also referred to as “licensing” or “registration” in this and other
documents (8).
National pandemic influenza preparedness plan: a national plan that
aims to set out country-specific priorities and actions, and to identify the major
components that must be put in place (for example, coordination, resource
identification and allocation, and capacity-building) along with response actions
that should be strengthened to respond to a pandemic (9).
Non-vaccine-producing country: a country in which vaccines are not
produced.
Pandemic influenza vaccine: a monovalent vaccine containing the
WHO Technical Report Series, No. 1004, 2017
human influenza A virus strain recommended by WHO for use either when a
pandemic is considered by WHO to be imminent or during a pandemic (1).
Pandemic phase: the period of global spread of human influenza
caused by a new virus strain. Progression from the interpandemic to the alert
and pandemic phases may occur quickly or gradually, as indicated by the global
risk assessment, principally based on virological, epidemiological and clinical
data (6).
Pandemic preparedness influenza vaccine: an influenza vaccine
developed and tested in anticipation of an influenza pandemic, and manufactured
using an influenza virus strain that is believed to have similar characteristics
to a potential pandemic virus strain (also referred to as “mock-up pandemic
influenza vaccine” or “vaccine against novel human influenza virus” in other
documents) (1, 10, 11).
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It should be noted that both joint reviews and the WHO collaborative
procedure require advance planning so that agreements are brought into effect
at the earliest opportunity and that the vaccine product is already identified.
It is expected that future pandemic influenza vaccines prequalified
by WHO will include a summary assessment report outlining the basis for
prequalification that will be available to countries intending to import, grant
marketing authorization for and use these vaccines to mitigate an influenza
pandemic. Requests for more detailed information regarding prequalification
of a particular pandemic influenza vaccine should be addressed to the WHO
prequalification programme.
The NRAs of some vaccine-producing countries with considerable
experience in the evaluation of seasonal and pandemic influenza vaccines
supported WHO in expediting the prequalification of pandemic influenza
vaccines during the 2009 H1N1 influenza pandemic, and are encouraged to
support the NRAs of non-vaccine-producing countries in regulatory decision-
making and marketing authorization of pandemic influenza vaccines.
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■■ the excipients in the candidate vaccine are the same as those in the
licensed vaccine; and
■■ the manufacturing technology (for example, eggs, inactivant,
purification process) and controls are the same as those of the
licensed vaccine.
Pandemic preparedness influenza vaccines are vaccines that have been
prepared using strains of influenza viruses that are considered to have pandemic
potential. These vaccines may be novel in formulation, antigen content and/or
adjuvant. Influenza vaccine manufacturers have been encouraged to develop
pandemic preparedness influenza vaccines and to conduct suitable nonclinical
and clinical testing to demonstrate their safety and immunogenicity.
The rationale for the decision to review pandemic preparedness influenza
vaccines should be made publicly available (10, 11).
Some countries may choose to make specific provisions for evaluating
pandemic preparedness influenza vaccine as a precautionary step so that the
strain-change policy and procedures used for seasonal influenza vaccine can be
adapted for suitable pandemic influenza vaccine applications. Once the pandemic
preparedness influenza vaccine has been evaluated and approved (although not
marketed for sale), the change to an appropriate pandemic virus strain – when
identified and formulated into a pandemic influenza vaccine – can be approved
using similar criteria to those used for an annual seasonal vaccine strain change.
This procedure may be implemented in countries with adequate regulatory
expertise and resources.
Some pandemic influenza vaccines or pandemic preparedness influenza
vaccines may be novel constructs or formulations requiring expert regulatory
evaluation. NRAs of non-vaccine-producing countries may request assistance in
such evaluations from WHO or other NRAs more experienced in the regulation
WHO Technical Report Series, No. 1004, 2017
of both seasonal and pandemic influenza vaccines (see section 4.3 above).
Where possible the NRA could make arrangements for the joint review
of pandemic influenza vaccine dossiers with neighbouring and/or supporting
NRAs. The possible parties involved in such an arrangement should establish
this agreement during the interpandemic phase.
Depending on the pandemic phase and the source of the vaccine,
review activities may include one or more of the following procedures (see also
Fig A7.1):
■■ full review;
■■ fast-track review of basic documentation;
■■ reliance;
■■ recognition;
■■ strain-change procedure.
5.2.3 Reliance
This is the process of reviewing the decisions of other competent NRAs with
which there has been an agreement for support. Where it has been agreed (as
defined in the approved NRA pandemic emergency procedures) that the decision
of another NRA can be considered and used as the basis of a recommendation
for marketing authorization, this approach would involve acceptance on the
basis of the already agreed conditions and limitations on the use of the vaccine,
and would require the following available documentation:
■■ certificate of the responsible NRA’s marketing authorization decision;
■■ assessment reports of the responsible NRA.
Applicability: this procedure would apply during the pandemic alert phase,
pandemic phase and transition phase for a pandemic influenza vaccine licensed
by an NRA other than a supporting NRA.
5.2.4 Recognition
This is the process of recognizing the WHO prequalification decision or
the decision of a supporting NRA. Where it has been agreed (as defined in
the approved NRA pandemic emergency procedures) that the decision of a
supporting NRA can be used as the basis for a recommendation for marketing
authorization, this approach would involve acceptance on the basis of the already
agreed conditions and limitations on the use of the vaccine, and would require
the following available documentation:
■■ certificate of the responsible NRA’s marketing authorization decision
or WHO prequalification assessment report.
Applicability: this procedure would apply during the pandemic alert phase,
pandemic phase and transition phase for a pandemic influenza vaccine licensed
by a supporting NRA or prequalified by WHO. It may also apply during the
pandemic phase for a pandemic influenza vaccine licensed by an NRA other
than a supporting NRA.
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WHO, the receiving NRA and the manufacturer should be signed in the
interpandemic phase – particularly given that, during an emergency, time may
not allow for this step to occur prior to the decision to use the vaccine. For this
procedure the WHO prequalification assessment report should be provided
to the receiving NRA. The full dossier in the format of the CTD could also be
provided to the NRA.
It would be expected that, following the pandemic phase, the full dossier
as required by the relevant non-vaccine-producing country would be completed
and submitted for evaluation.
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Fig. A7.1
Illustrative chart of regulatory approaches relative to the status of the vaccine and the
continuum of pandemic phases a
Recognition in the
pandemic phase
Recognition Recognition OR
procedure in all procedure in all Reliance or Fast-
phases phases track procedure in
the alert or transition
phases
a
Interpandemic phase, alert phase, pandemic phase or transition phase as defined by WHO (see section 3,
Terminology above).
b
Any NRA selected by the NRA of the receiving country as suitable in supporting pandemic influenza vaccine
licensing decisions; the eligibility of supporting NRAs could be decided upon after consultation with WHO.
c
Any NRA not designated as a “supporting NRA” by the NRA of the receiving country.
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■■ There is a local agency responsible for supply of the product (that is,
an “applicant” or state body that is a defined responsible legal entity).
■■ Packaging, label and package insert are nationally acceptable.
■■ The vaccine is compatible with the national pandemic influenza
preparedness plan.
■■ The vaccine is indicated for the circulating strain(s).
This evaluation may need to be based upon minimal and incomplete
documentation, and this should be acknowledged in the recommendation.
An evaluation report should be produced by the NRA.
References
1. Guidelines on regulatory preparedness for human pandemic influenza vaccines. In: WHO Expert
Committee on Biological Standardization: fifty-eighth report. Geneva: World Health Organization;
2011: Annex 2 (WHO Technical Report Series, No. 963; http://www.who.int/biologicals/vaccines/
Annex_2_WHO_TRS_963-3.pdf?ua=1, accessed 28 November 2016).
2. Workshop on international regulatory capacity enhancement for influenza vaccines, 8–10 June
2011, São Paulo, Brazil. Meeting report. Geneva: World Health Organization; 2011 (http://www.
who.int/immunization_standards/national_regulatory_authorities/wirceiv_report_18jan2012.
pdf, accessed 28 November 2016).
3. Main operational lessons learnt from the WHO pandemic influenza A(H1N1) vaccine deployment
initiative. Report of a WHO meeting held in Geneva, Switzerland, 13–15 December 2010. Geneva:
World Health Organization; 2011 (http://www.who.int/influenza_vaccines_plan/resources/h1n1_
vaccine_deployment_initiaitve_moll.pdf, accessed 28 November 2016).
4. Pandemic influenza preparedness and response: a WHO guidance document. Geneva: World
Health Organization; 2009 (reprinted 2010) (http://apps.who.int/iris/bitstream/10665/44123/1/
9789241547680_eng.pdf, accessed 28 November 2016).
5. Pandemic Influenza Preparedness Framework. Partnership contribution implementation plan
2013–2016. Geneva: World Health Organization; 2013 (http://www.who.int/influenza/pip/pip_
pcimpplan_17jan2014.pdf, accessed 28 November 2016).
479
WHO Expert Committee on Biological Standardization Sixty-seventh report
6. Pandemic Influenza Risk Management: WHO interim guidance. Geneva: World Health
Organization; 2013 (http://www.who.int/influenza/preparedness/pandemic/GIP_Pandemic
InfluenzaRiskManagementInterimGuidance_Jun2013.pdf?ua=1, accessed 28 November 2016).
7. International Health Regulations (2005). Second edition. Geneva: World Health Organization;
2008 (reprinted 2008) (http://apps.who.int/iris/bitstream/10665/43883/1/9789241580410_eng.
pdf, accessed 28 November 2016).
8. Guidelines on procedures and data requirements for changes to approved vaccines. In: WHO
Expert Committee on Biological Standardization: sixty-fifth report. Geneva: World Health
Organization; 2015: Annex 4 (WHO Technical Report Series, No. 993; http://www.who.int/
biologicals/vaccines/Annex4_Guidelines_changes_to_approved_vaccines_eng.pdf?ua=1,
accessed 28 November 2016).
9. Comparative analysis of national pandemic influenza preparedness plans. January 2011.
Geneva: World Health Organization; 2011 (http://www.who.int/influenza/resources/documents/
comparative_analysis_php_2011_en.pdf?ua=1, accessed 28 November 2016).
10. Pandemic influenza A(H1N1)v vaccines authorised via the core dossier procedure. Explanatory
note on scientific considerations regarding the licensing of pandemic A(H1N1)v vaccines. London:
European Medicines Agency; 2009 (EMEA/608259/2009 rev.; http://www.ema.europa.eu/docs/en_
GB/document_library/Medicine_QA/2009/11/WC500007567.pdf, accessed 28 November 2016).
11. Pandemic report and lessons learned. Outcome of the European Medicines Agency’s
activities during the 2009 (H1N1) flu pandemic. London: European Medicines Agency; 2011
(EMA/221017/2011; http://www.ema.europa.eu/docs/en_GB/document_library/Report/2011/04/
WC500105820.pdf, accessed 28 November 2016).
12. Guidance on development and implementation of a national deployment and vaccination plan
for pandemic influenza vaccines. Geneva: World Health Organization; 2012 (http://apps.who.int/
iris/bitstream/10665/75246/1/9789241503990_eng.pdf, accessed 28 November 2016).
13. WHO checklist for influenza pandemic preparedness planning. Geneva: World Health
Organization; 2005 (WHO/CDS/CSR/GIP/2005.4; http://www.who.int/influenza/resources/
documents/FluCheck6web.pdf?ua=1, accessed 28 November 2016).
14. WHO outbreak communication guidelines. Geneva: World Health Organization; 2005
(WHO/CDS/2005.28.) (http://apps.who.int/iris/bitstream/10665/69369/1/WHO_CDS_2005_28_
eng.pdf?ua=1, accessed 28 November 2016).
15. World Health Organization outbreak communication planning guide. 2008 edition. Geneva: World
WHO Technical Report Series, No. 1004, 2017
19. Procedure for assessing the acceptability, in principle, of vaccines for purchase by United Nations
agencies. In: WHO Expert Committee on Biological Standardization: sixty-first report. Geneva:
World Health Organization; 2013: Annex 6 (WHO Technical Report Series, No. 978; http://www.
who.int/immunization_standards/vaccine_quality/TRS_978_61st_report_Annex_6_PQ_vaccine_
procedure.pdf?ua=1, accessed 27 March 2017).
20. Guidelines for medicine donations. Revised 2010. Third edition 2011. Geneva: World Health
Organization; 2011 (http://apps.who.int/iris/bitstream/10665/44647/1/9789241501989_eng.pdf,
accessed 28 November 2016).
21. Guidance for industry. Expedited programs for serious conditions – drugs and biologics. Silver
Spring (MD): Food and Drug Administration; 2014 (http://www.fda.gov/downloads/drugs/
guidancecomplianceregulatoryinformation/guidances/ucm358301.pdf, accessed 28 November
2016).
22. Regulation and licensing of biological products in countries with newly developing regulatory
authorities. In: WHO Expert Committee on Biological Standardization: forty-fifth report. Geneva:
World Health Organization; 1995: Annex 1 (WHO Technical Report Series, No. 858; http://www.
who.int/biologicals/publications/trs/areas/biological_therapeutics/WHO_TRS_858_A1.pdf?ua=1,
accessed 28 November 2016).
23. Regulation of vaccines: building on existing drug regulatory authorities. Geneva: World
Health Organization; 1999 (WHO/V&B/99.10; http://apps.who.int/iris/bitstream/10665/65968/1/
WHO_V-B_99.10_eng.pdf, accessed 28 November 2016).
24. Guidelines for national authorities on quality assurance for biological products. In: WHO
Expert Committee on Biological Standardization: forty-second report. Geneva: World Health
Organization; 1992: Annex 2 (WHO Technical Report Series, No. 822; http://www.who.int/
biologicals/publications/trs/areas/biological_therapeutics/WHO_TRS_822_A2.pdf?ua=1, accessed
28 November 2016).
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Appendix 1
Checklist of regulatory actions for pandemic influenza
preparedness and response
It is important to ensure that regulatory legislation is in place to enable the
various approaches listed below to be applied as needed in preparation for, or
during, a pandemic.
1. Prepare regulatory preparedness procedures compatible with the national
pandemic influenza preparedness plan during the interpandemic phase.
2. Appoint and maintain a pandemic task team (with staff, training, budget and
annual review).
3. In the interpandemic phase (provisionally) grant marketing authorization to
pandemic preparedness influenza vaccines.
4. Liaise with other national agencies on pandemic preparedness procedures.
5. Develop memoranda of understanding with potential supporting NRAs.
6. In the pandemic alert phase (or earlier, if possible):
(a) determine which vaccines may be sourced by national agencies;
(b) request data packages from potential vaccine suppliers;
(c) decide on appropriate evaluation procedures and evaluators;
(d) prepare a format for an assessment report and post-marketing
surveillance plan;
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Appendix 2
Examples of information and documentation that may
be required for the evaluation of a seasonal influenza
vaccine annual virus strain change
1. WHO-recommended strain list for the relevant hemisphere.
2. Manufacturer’s choice of strains for inclusion.
3. Details of manufacturing procedure (declaration if unchanged).
4. Validation of the inactivation and fragmentation.
5. Source, history and master/working seed characterization of each strain
included.
6. Egg or cell culture: safety specifications and tests (declaration if unchanged).
7. Qualification of potency test (single radial immunodiffusion – SRID)
reagents.
8. Final product release specifications and results (this must include endotoxin
release limit).
9. Retrospective data on the “efficacy or performance” of influenza vaccines
(preceding year or season).
10. Stability data (accelerated or from the most recent, or most similar, batch of
approved vaccine).
WHO Technical Report Series, No. 1004, 2017
484
Annex 7
485
Annex 8
Labelling information of inactivated influenza vaccines
for use in pregnant women
Addendum to Annex 3 of WHO Technical Report Series, No. 927
1. Introduction 490
2. Background 490
3. Purpose and scope 492
4. Terminology 492
5. Labelling information 493
5.1 Indications and Usage 493
5.2 Warnings and Precautions 494
5.3 Contraindications 495
5.4 Use in Specific Populations 495
6. Summary 497
Authors and acknowledgements 497
References 499
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Abbreviations
EMA European Medicines Agency
GACVS WHO Global Advisory Committee on Vaccine Safety
IFPMA International Federation of Pharmaceutical Manufacturers
& Associations
IIV inactivated influenza vaccine
NITAG national immunization technical advisory group
NRA national regulatory authority
SAGE WHO Strategic Advisory Group of Experts
SmPC summary of product characteristics
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1. Introduction
Rates of morbidity and mortality resulting from seasonal influenza virus
infection are considered to be substantial worldwide (1, 2). Pregnant women
are especially vulnerable and have an increased risk of severe disease and
death from influenza. The infection may also lead to fetal complications such
as stillbirth, neonatal death, preterm delivery and decreased birthweight
(3, 4). For these reasons, the 2012 WHO position paper on vaccines against
influenza (3) – endorsed by the WHO Strategic Advisory Group of Experts on
Immunization (SAGE) – recommended the immunization of pregnant women
with trivalent inactivated influenza vaccine (IIV) at any stage of pregnancy.
SAGE also recommended that pregnant women should be given the highest
priority in countries considering the initiation or expansion of immunization
programmes for seasonal influenza vaccination (3, 5, 6). This recommendation
is based on evidence of a substantial risk of severe disease in this population
group and on evidence that the use of seasonal influenza vaccine is both safe
throughout pregnancy and effective in preventing influenza in women as well
as in their young infants in whom the disease burden is also high (3, 5). After
careful analysis of data worldwide, the WHO Global Advisory Committee
on Vaccine Safety (GACVS) concluded that there was no evidence of adverse
pregnancy outcomes associated with the vaccination of pregnant women with
several inactivated viral or bacterial vaccines, including IIVs (5, 6). However,
for various reasons, the implementation of influenza immunization during
pregnancy remains suboptimal (4). One reason for this has been the perceived
risk of administering influenza vaccine, or indeed any vaccine, to this population
group, particularly due to the precautionary language used in some product
labels and the likelihood of misinterpretation (7).
The development of this explanatory addendum arises from the
WHO Technical Report Series, No. 1004, 2017
2. Background
Enhancing the uptake of vaccines during pregnancy is an important element
of WHO’s ongoing work to improve maternal and child health. As part of this
work, WHO held a consultation in July 2014 on influenza vaccines for pregnant
and lactating women which focused on the clinical data requirements for
product labelling information (8). The consultation was organized by the WHO
Technologies, Standards and Norms team and the WHO Initiative for Vaccine
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4. Terminology
The definitions given below apply to the terms as used in this WHO guidance
document. These terms may have different meanings in other contexts.
Label(ling): all forms of product information – that is, container label,
SmPC, product/package insert, package leaflet and prescribing information.
Maternal immunization: frequently used to refer to vaccination prior
to, during or after pregnancy. For the purposes of this document the term refers
specifically to vaccination during pregnancy.
National Immunization Technical Advisory Group (NITAG): a national
expert advisory group that evaluates the available evidence on national disease
incidence, and available vaccines, in order to provide advice to the health ministry
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5. Labelling information
As with all prescription drugs and biological products, IIVs must be accompanied
by labelling that summarizes the scientific information concerning their safe and
effective use.
Labelling includes the package insert, which is also referred to as
prescribing information or the SmPC (17). This component of labelling is
the primary mechanism through which regulatory agencies and vaccine
manufacturers communicate essential, science-based information. This
information is then used by health-care professionals to make prescribing
decisions and to counsel patients about the risks and benefits of a product. The
content and format requirements for labelling are prescribed by regulations
specific to the country where the vaccine is licensed and may differ between
countries (17, 18). Nevertheless, common principles include that prescribing
information should be based on available data, that it must not be misleading
and that it must not contain implied claims or uses for which there is inadequate
evidence of safety or effectiveness (19).
The labelling sections relevant to the use of vaccines in pregnancy –
namely, “Indications and Usage”, “Warnings and Precautions”, “Contraindications”,
and “Use in Specific Populations” – are described below. Countries have
information on vaccination in pregnancy under various sections. Information
regarding the use of an IIV in pregnancy is typically found under the “Use in
Specific Populations” section. However, in some countries the NRA has required
that precautionary statements about the use of an IIV in pregnancy should be
included under the “Warnings and Precautions” and “Contraindications” sections
because safety data on use of the vaccine in pregnancy may be unavailable
or insufficient.
5.3 Contraindications
Although the specific wording used in the “Contraindications” section of
the product labelling may depend on the requirements of the NRA where
the vaccine has been licensed, there is a common requirement that drugs
or biological products, including vaccines, should be contraindicated only
in those situations where the known risk from use clearly outweighs any
possible benefit. Only known hazards, not theoretical possibilities, should
be the basis for contraindication. As an example relating to vaccine use in
pregnant women, evidence in humans or animals that a vaccine poses a serious
risk of developmental toxicity during pregnancy would usually warrant a
contraindication for use during pregnancy. However, for IIVs, if available
animal or human data do not indicate a risk in pregnancy that clearly outweighs
benefit, or if data are unavailable to inform risk in pregnancy, there should not
be a contraindication for use during pregnancy.
Data that may be available concerning the safety of the product in pregnant
women are also described in this section. Sources of human data may include
pregnancy registries, pre-licensure clinical trials in which pregnant women were
inadvertently exposed to the product, large-scale epidemiological studies and
case-series reporting rare adverse events. In general, information regarding use of
IIVs during pregnancy is derived from post-marketing studies (for example, via
registries and/or from maternal immunization studies published in the literature).
The quality and quantity of data from specific sources will be evaluated by the
NRA to determine whether the data are scientifically acceptable for inclusion in
the pregnancy subsection of the product labelling. In some countries, the NITAG
recommendations are included in this section.
As with other sections of the product labelling, country-specific
requirements prescribe the information, and frequently the specific wording, to
be included in the pregnancy subsection in relation to what is known about the
risks of using the product in pregnancy.
WHO’s prequalification evaluation of the prescribing information
is evidence-based and takes into consideration the prescribing information
approved by the NRA of record for prequalification (generally the NRA in the
country of manufacture).
Required statements included in the pregnancy subsection of the
product labelling have often been precautionary (for example, Should be used
only following advice of a health-care professional; If pregnant, please inform your
doctor or pharmacist; Use only if clearly needed). The rationale for requiring
such language has largely stemmed from a lack of data from well-controlled
clinical trials rather than evidence suggesting specific risks of vaccination during
pregnancy. Such precautionary language has sometimes been misinterpreted to
mean that pregnancy is a contraindication for use.
Whereas many NRAs require that labelling includes such precautionary
language regarding use in pregnancy, some countries are considering ways to
WHO Technical Report Series, No. 1004, 2017
6. Summary
IIVs are not contraindicated for use in pregnancy. The “Indications and Usage”
section for these vaccines specifies an age range that includes women of
childbearing age. Consequently, the lack of an “Indications and Usage” statement
specifically addressing use in pregnant women does not preclude use of these
vaccines during pregnancy. Certain countries include information on the use of
IIV in pregnancy under the “Warnings and Precautions” or “Contraindications”
sections of product labelling. However, this does not reflect a known or
suspected safety issue relating to the use of these vaccines during pregnancy
but rather a precautionary approach taken by certain NRAs. Programmatic
recommendations (such as those from SAGE and some NITAGs) on the use of
IIVs during pregnancy are consistent with labelling.
References
1. Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V et al. Global and regional mortality
from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global
Burden of Disease Study 2010. Lancet. 2012;380:2095–128 (abstract: http://www.thelancet.com/
journals/lancet/article/PIIS0140-6736(12)61728-0/abstract, accessed 5 December 2016).
2. Zhou H, Thompson WW, Viboud CG, Ringholz CM, Cheng PY, Steiner C et al. Hospitalizations
associated with influenza and respiratory syncytial virus in the United States, 1993–2008. Clin
Infect Dis. 2012;54(10):1427–36 (abstract: https://www.ncbi.nlm.nih.gov/pubmed/22495079,
accessed 5 December 2016).
3. Vaccines against influenza. WHO position paper – November 2012. Wkly Epidemiol Rec.
2012;87(47):461–76 (http://www.who.int/wer/2012/wer8747.pdf?ua=1, accessed 5 December
2016).
4. Meeting of the Strategic Advisory Group of Experts on immunization, November 2013 –
conclusions and recommendations. Wkly Epidemiol Rec. 2014;89(1):1–20 (http://www.who.int/
wer/2014/wer8901.pdf?ua=1, accessed 6 December 2016).
5. Report on safety of immunization during pregnancy. WHO Global Advisory Committee on
Vaccine Safety, 18 October 2013. Geneva: World Health Organization; 2013 (http://www.who.
int/immunization/sage/meetings/2013/november/2_GACVS_pregnancy_report.pdf, accessed 6
December 2016).
6. Global Advisory Committee on Vaccine Safety, 12–13 June 2013. Wkly Epidemiol Rec.
2013;88(29):301–12 (http://www.who.int/wer/2013/wer8829.pdf, accessed 6 December 2016).
499
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7. Top KA, Arkell C, Scott H, McNeil SA, Mannerfeldt J, Ortiz JR et al. Effect of package insert
language on health-care providers’ perceptions of influenza vaccination safety during pregnancy.
Lancet. 2016;4(10):e690–1 (http://www.thelancet.com/journals/langlo/article/PIIS2214-109X(16)
30182-6/fulltext, accessed 6 December 2016).
8. WHO consultation on influenza vaccines for pregnant and lactating women: clinical data
requirements for product labelling, 15–16 July 2014, Geneva, Switzerland. Executive summary.
Geneva: World Health Organization; 2014 (http://www.who.int/biologicals/vaccines/INFLUENZA_
VACCINES_Executive_Summary_FINAL_29_Sept_R.pdf, accessed 6 December 2016).
9. WHO working group meeting on labelling information of influenza vaccines intended to be used
in pregnant women, 24–25 September 2015, Geneva, Switzerland. Executive summary. Geneva:
World Health Organization; 2015 (http://www.who.int/biologicals/areas/vaccines/Executive_
summary_FINAL_labelling_Sept_mtg_16_Dec_2015.pdf, accessed 6 December 2016).
10. WHO informal consultation on labelling information of influenza vaccines intended to be
used in pregnant women, 4–5 April 2016, Geneva, Switzerland. Executive summary. Geneva:
World Health Organization; 2016 (http://www.who.int/biologicals/areas/vaccines/INFLUENZA_
Executive_summary_Final_labelling_Apri_mgt_10_June_2016.pdf?ua=1, accessed 6 December
2016).
11. Labelling information for influenza vaccines intended for use in pregnant women. In: WHO
Expert Committee on Biological Standardization: sixty-sixth report. Geneva: World Health
Organization; 2016:25–26 (WHO Technical Report Series, No. 999; http://apps.who.int/iris/bitstr
eam/10665/208900/1/9789240695634-eng.pdf?ua=1, accessed 6 December 2016).
12. Recommendations for the production and control of influenza vaccine (inactivated). In: WHO
Expert Committee on Biological Standardization: fifty-fourth report. Geneva: World Health
Organization; 2005: Annex 3 (WHO Technical Report Series, No. 927; http://www.who.int/
biologicals/publications/trs/areas/vaccines/influenza/ANNEX%203%20InfluenzaP99-134.pdf,
accessed 6 December 2016).
13. Keller-Stanislawski B, Englund JA, Kang G, Mangtani P, Neuzil K, Nohynek H et al. Safety of
immunization during pregnancy: a review of the evidence of selected inactivated and live
attenuated vaccines. Vaccine. 2014;32(52):7057–64 (abstract: https://www.ncbi.nlm.nih.gov/
pubmed/25285883/, accessed 6 December 2016).
14. Guideline on influenza vaccines. Non-clinical and clinical module. London: European Medicines
Agency; 2014 (EMA/CHMP/VWP/457259/2014; http://www.ema.europa.eu/docs/en_GB/
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19. Marshall V, Gruber M. Influenza immunization during pregnancy: US regulatory perspective. Am J
Obstet Gynecol. 2012;207(3 Suppl):S57–62 (http://www.ajog.org/article/S0002-9378(12)00729-
6/pdf, accessed 6 December 2016).
20. Roberts JN, Gruber MF. Regulatory considerations in the clinical development of vaccines
indicated for use during pregnancy. Vaccine. 2015;33(8):966–72 (abstract: http://www.
sciencedirect.com/science/article/pii/S0264410X1401723X, accessed 6 December 2016).
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Guidelines on clinical evaluation of vaccines: regulatory
expectations
Replacement of Annex 1 of WHO Technical Report Series, No. 924
1. Introduction 506
2. Purpose and scope 508
3. Terminology 510
4. Vaccine clinical development programmes 514
4.1 General considerations 514
4.2 Pre-licensure clinical development programmes 516
4.3 Post-licensure clinical evaluations 517
5. Immunogenicity 518
5.1 General considerations 518
5.2 Characterization of the immune response 518
5.3 Measuring the immune response 519
5.4 Identification and use of immune correlates of protection 524
5.5 Immunogenicity trials 526
5.6 Specific uses of immunogenicity trials 531
6. Efficacy and effectiveness 545
6.1 General considerations for efficacy trials 545
6.2 Types of efficacy trials 547
6.3 Design and conduct of efficacy trials 548
6.4 Approaches to determination of effectiveness 558
7. Safety 560
7.1 General considerations 560
7.2 Assessment of safety in clinical trials 560
7.3 Size of the pre-licensure safety database 566
7.4 Post-licensure safety surveillance 567
Authors and acknowledgements 569
References 571
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Abbreviations
AE adverse event
AEFI adverse event following immunization
AESI adverse event of special interest
AR attack rate
ARU attack rate in unvaccinated (control group)
ARV attack rate in vaccinated group
DNA deoxyribonucleic acid
ELISA enzyme-linked immunosorbent assay
GCP good clinical practice
GMC geometric mean concentration
GMP good manufacturing practice
GMT geometric mean titre
HPV human papillomavirus
ICH International Conference on Harmonisation of Technical
Requirements for Registration of Pharmaceuticals for
Human Use
ICP immune correlate of protection
IgG immunoglobulin G
LLOD lower limit of detection
LLOQ lower limit of quantification
NRA national regulatory authority
OPA opsonophagocytic antibody
RNA ribonucleic acid
RR relative risk
SAE serious adverse event
SBA serum bactericidal antibody
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1. Introduction
These WHO Guidelines are intended to replace the WHO Guidelines on clinical
evaluation of vaccines: regulatory expectations, which were adopted by the Expert
Committee on Biological Standardization in 2001 (1). The document of 2001
provided guidance on the clinical evaluation of vaccines as well as on WHO
vaccine prequalification.
Since 2001, more than 20 vaccine-specific documents (each including a
section on clinical evaluation) have been adopted by the Committee. Originally
intended to be read in conjunction with the 2001 document, these documents
provide guidance on both oral and inactivated polio vaccines, whole cell pertussis
and acellular pertussis vaccines, meningococcal conjugate vaccines for serotypes
A and C, and pneumococcal conjugate vaccines, as well as on vaccines intended to
prevent diseases caused by rotaviruses, dengue viruses, human papillomaviruses
(HPVs) and malaria parasites.
These revised WHO Guidelines have been prepared to reflect the
scientific and regulatory experience that has been gained from vaccine clinical
development programmes since the adoption of the 2001 version. They are
intended for use by national regulatory authorities (NRAs), companies developing
and holding licences for vaccines, clinical researchers and investigators. The
document takes into account the content of clinical development programmes,
clinical trial designs, the interpretation of trial results and post-licensing activities.
The main content changes (modification or expansion of previous text
and additional issues covered) include, but are not limited to, the following:
Immunogenicity
■■ general principles for comparative immunogenicity studies,
including selection of the comparators, end-points and acceptance
WHO Technical Report Series, No. 1004, 2017
Safety
■■ detailed consideration of the collection and analysis of safety data
from clinical trials;
■■ consideration of size of the pre-licensure database by type of vaccine
and its novelty;
■■ consideration of the safety database by population subgroup;
■■ special safety considerations by vaccine construct;
■■ circumstances of limited pre-licensure safety data;
■■ use of registries;
■■ issues regarding vaccine pharmacovigilance activities.
Because a separate document on the nonclinical evaluation of vaccines
was established in 2003 (2), the corresponding section in the 2001 Guidelines has
been removed. Furthermore, the structure of the document has changed, with
a number of methodological considerations now incorporated into the relevant
sections and subsections rather than being described in a separate section. In
line with all the changes made in the document, the terminology and references
have been updated.
WHO has also made available several guidelines, manuals and reports
relevant to vaccine clinical development programmes. These should be consulted
as appropriate, and include:
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States Food and Drug Administration and the United Kingdom Medical
Research Council. These WHO Guidelines are intended to complement these
other documents.
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may be an acute infectious disease and/or a disease that results from chronic
infection with an infectious agent.
These Guidelines are applicable to the clinical development of:
■■ new candidate vaccines;
■■ licensed vaccines;
■■ vaccines that are given by any route of administration;
■■ vaccines that may be given before exposure or shortly after known
or presumed exposure to an infectious agent to prevent the onset of
clinical disease.
The Guidelines are further applicable to vaccines that contain one or
more of the following:
■■ microorganisms that have been inactivated by chemical and/or
physical means;
■■ live microorganisms that are not virulent in humans as a result of
attenuation processes or specific genetic modification;
■■ antigenic substances that have been derived from microorganisms
(these may be purified from microorganisms and used in their
natural state, or they may be modified, for example, detoxified by
chemical or physical means, aggregated or polymerized);
■■ antigens that have been manufactured by synthetic processes or
produced by live organisms using recombinant RNA or DNA
technology;
■■ antigens (however manufactured) that have been chemically
conjugated to a carrier molecule to modify the interaction of the
antigen with the host immune system;
■■ antigens that are expressed by another microorganism which itself
does not cause clinical disease but acts as a live vector (for example,
live viral vectored vaccines and live-attenuated chimeric vaccines).
In addition, although naked DNA vaccines are not specifically discussed,
the principles and development programmes outlined are broadly applicable.
These Guidelines do not apply to:
■■ therapeutic vaccines (that is, those intended for treatment
of disease);
■■ vaccines intended for any purpose other than the prevention of
clinical disease caused by infectious agents.
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3. Terminology
The definitions given below apply to the terms as used in these WHO Guidelines.
These terms may have different meanings in other contexts.
Adverse event (AE): any untoward medical occurrence in a participant
in a clinical trial. An AE does not necessarily have a causal relationship with
the vaccine.
Adverse event following immunization (AEFI): any untoward medical
occurrence that follows immunization and which does not necessarily have a
causal relationship with the use of the vaccine. The AEFI may be any unfavourable
or unintended sign, abnormal laboratory finding, symptom or disease. In clinical
trial documentation AEFI may often be shortened to AE.
Adverse event of special interest (AESI): a clinically important untoward
medical occurrence that is either known to occur following administration of
the type of vaccine under study (for example, hypotonic-hyporesponsive
episodes or febrile convulsions) or is considered to be a possible risk on the
basis of knowledge of the content of the vaccine and/or its interaction with the
host immune system (for example, autoimmune disease or antibody-dependent
enhanced clinical disease).
Attack rate (AR): the proportion of the population exposed to an
infectious agent that goes on to develop clinically manifest disease.
Blinding: a procedure by which one or more parties involved in a
clinical trial are kept unaware of the randomized intervention.
Booster dose: a dose that is given at a certain interval after completion of
the primary series that is intended to boost immunity to, and therefore prolong
protection against, the disease that is to be prevented.
Case ascertainment: the method adopted for detecting cases of the
disease targeted for prevention by vaccination in a vaccine efficacy trial or in a
study of vaccine effectiveness.
WHO Technical Report Series, No. 1004, 2017
assay. This term may be applied whether or not there is an established ICP and
when the clinical relevance of achieving or exceeding the predefined response
is unknown.
Responder rate: the responder rate is the percentage of subjects in a
treatment group with immune responses that meet (or exceed) a predefined
immune response.
Serious adverse event (SAE): an AE is serious when it results in: (a)
death, admission to hospital, prolongation of a hospital stay, persistent or
significant disability or incapacity; (b) is otherwise life-threatening; or (c) results
in a congenital abnormality or birth defect. Some NRAs may have additional or
alternative criteria for defining SAEs.
Seroconversion: a predefined increase in serum antibody concentration
or titre. In subjects with no detectable antibody – below the lower limit of
detection (LLOD) – or no quantifiable antibody – below the lower limit of
quantification (LLOQ) – prior to vaccination, seroconversion is usually defined
as achieving a quantifiable antibody level post-vaccination. In subjects with
quantifiable antibody prior to vaccination, seroconversion is commonly defined
by a predefined fold-increase from pre- to post-vaccination.
Sponsor: the individual, company, institution or organization that takes
responsibility for the initiation, management and conduct of a clinical trial. The
sponsor of a clinical trial may not be the entity that applies for a licence to place
the same product on the market or the entity that holds the licence (that is, is
responsible for post-licensing safety reporting) in any one jurisdiction.
Superiority trial: a trial with the primary objective of demonstrating
that a test group is superior to a reference group on the basis of the primary
end-point. In the context of vaccine development the primary end-point may
be a safety parameter (for example, occurrence of a specific type of AE), a
clinical condition (for example, occurrence of a specific infectious disease) or an
immunological parameter (for example, a measure of the immune response to
one or more antigenic components of the vaccine).
Vaccine efficacy: vaccine efficacy measures direct protection (that is,
protection induced by vaccination in the vaccinated population sample). Vaccine
efficacy is most commonly a measure of the proportionate reduction in disease
attack rate (AR) between the control group that did not receive vaccination
against the infectious disease under study (ARU) and the vaccinated (ARV)
group(s). Vaccine efficacy can be calculated from the relative risk (RR) of disease
among the vaccinated group as (ARU − ARV/ARU) × 100 and (1 − RR) × 100.
This estimate may be referred to as absolute vaccine efficacy. Alternatively,
vaccine efficacy may be defined as a measure of the proportionate reduction
in disease AR between a control group that is vaccinated against the infectious
disease under study and the group vaccinated with the candidate vaccine. This
estimate may be referred to as relative vaccine efficacy.
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The key outcomes of a clinical trial should be posted in the results section
of the entry in the clinical trial registry and/or posted on a publicly available,
free to access and searchable website (for example, that of the trial sponsor or
principal investigator). It is recommended that posting of these results should
usually occur within 12 months of completion or termination of the study, or in
accordance with the relevant NRA requirements.
Depending on individual NRA requirements, some or all regulatory
submissions may need to include a listing of all completed and ongoing trials
conducted with the vaccine by the sponsor. It is recommended that any trials
that are known to the sponsor (for example, from searching registries or from
publications) that were initiated by entities other than the sponsor (for example,
by a public health body, academic institution or another company that used the
product as a comparator) should be included.
are conducted in healthy adults. It may be appropriate, if feasible, that the first
trials are confined to subjects who have no history of previous exposure to the
organism(s) against which the candidate vaccine is intended to protect.
Further safety and immunogenicity trials that are conducted to build on
the Phase I trial results are commonly referred to as Phase II trials. In most cases
these trials are conducted in subjects who are representative of the intended
target population for the vaccine at the time of licensure. For vaccines intended
for a broad age range it may not be necessary in all instances to apply an age
de-escalation approach (for example, to move from adults to adolescents, then
to children aged 6–12 years, followed by younger children, toddlers and finally
infants) to sequential trials or to groups within trials. For example, if a vaccine
has negligible potential benefit for older children it may be acceptable in some
cases to proceed directly from trials in adults to trials in younger children,
including infants and toddlers.
These trials are usually designed to provide sufficient safety and
immunogenicity data to support the selection of one or more candidate
formulations for evaluation in pivotal trials (that is, to select the amount(s) of
antigenic component(s) and, where applicable, adjuvant in each dose).
5. Immunogenicity
5.1 General considerations
Immunogenicity trials are conducted at all stages of pre-licensure vaccine
development and additional trials may be conducted in the post-licensure
period. The evaluation of immune responses relies upon the collection of
adequate specimens at appropriate time intervals and the measurement of
immune parameters most relevant to the vaccine.
Pre-licensure and post-licensure clinical trials commonly evaluate and
compare immune responses between trial groups to address a range of objectives.
In trials that are primarily intended to estimate vaccine efficacy and/or safety,
assessment of the immune response is usually a secondary objective but it is
important that data on immune responses are collected to support analyses of
the relationship between immunogenicity and efficacy, which may lead to the
identification of ICPs.
those that measure the humoral immune response have not played a pivotal
or major role in vaccine licensure, so the focus is usually on determination of
antibody levels.
■■ For known microorganisms or antigens in a candidate vaccine the
range of parameters to be measured in clinical trials is usually
selected on the basis of prior experience and whether or not there is
an established ICP.
■■ For microorganisms or antigens not previously included in human
vaccines the selection of parameters to be measured should take into
account what is known about natural immunity. For some infectious
diseases the nature of the immune response to infection in animal
models may also be useful for parameter selection.
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5.3.3 Assays
Assays of functional or total antibody that are used to report immune responses
to vaccination (whether to the candidate vaccine or to co-administered vaccines)
in trials intended to support licensure (that is, in pivotal trials) should be
acceptable to the relevant NRAs. They may be:
■■ commercially available assays specifically designed and intended
for quantification of antibody (that is, assays that have undergone a
robust regulatory review);
■■ assays that are not commercially available but have been validated
according to principles similar to those recommended for
quantitative lot release assays in the ICH Q2 (R1) document
Validation of analytical procedures: text and methodology (16);
■■ assays that are not commercially available but have been shown to be
comparable to a reference assay (for example, to an assay established
in a WHO reference laboratory or to an assay that is established in a
recognized public health laboratory and has been used previously to
support clinical trials that were pivotal for licensure).
It is expected that, if these exist, WHO International Standards and
Reference Reagents will be used in assay runs. Any omission of their use should
be adequately justified.
Clinical trial protocols should specify which assays will be used.
Clinical trial reports should include a summary of the assay methodology and
its commercial or other validation status. For assays that are not commercially
available any available validation reports should be provided.
The same assays should preferably be used in the same laboratories
throughout the clinical development programme (including pre- and post-
licensure trials) for an individual vaccine. It is also preferable that each assay
(whether it measures the response to the candidate vaccine or to a concomitant
vaccine) is run by one central laboratory. If this is not possible (for example,
because different laboratories have to be used, assays change over time, or a
switch is made to an improved and/or more suitable assay) the new and original
assays should be shown to give the same result or interpretation, or the impact
of any differences should be evaluated and the use of a new assay justified. It
is recommended that, as a minimum, a selection of stored sera (for example,
covering a range of low to high results when using the previous assay) should be
re-run using the previous and new assays in parallel. The number of sera retested
should be sufficient to support a statistical assessment of assay comparability
and/or reproducibility.
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5.4
5.4.1 Immune correlates of protection and their uses
All established ICPs are based on humoral immune response parameters that
measure functional or total IgG antibody. Some examples of well-established
ICPs include those for antibody to diphtheria and tetanus toxoids, polioviruses,
hepatitis B virus and Haemophilus influenzae type b capsular polysaccharide (17).
In most cases established ICPs have been shown to correlate with prevention
of clinically apparent infectious disease, but for some pathogens the ICP \
correlates with prevention of documented infection (for example, hepatitis A and
hepatitis B).
Sections 5.5.2 and 5.5.3 below consider trial end-points and the approach
to analysis and interpretation of immunogenicity data in the presence or absence
of an ICP.
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intended to prevent the same infectious disease. In addition, an ICP may not
be applicable to other populations and disease settings. For example, putative
ICPs have sometimes differed between populations of different ethnicities with
variable natural exposure histories for subtypes of a single microorganism. Thus,
the reliance that is placed on a clinical ICP, even if regarded as well supported
by the evidence, should take into account details of the efficacy trials from which
it was derived.
Clinical ICPs have also been derived from, or further supported by,
data collected during use of a vaccine to control an outbreak and from analyses
of effectiveness data. The methods used to derive ICPs from these types of
data have been very variable. The estimates may in part reflect the type of
immunization programme put in place and the extent to which the protection
of individuals relies on herd immunity rather than the initial and persisting
immune response in the individual. Therefore the wider applicability of ICPs
derived from interventional or routine use should be viewed in the light of how
and in what setting the estimates were obtained.
If it is not possible to derive a clinical ICP the interpretation of the
human immune response data may take into account what is known about
immunological parameters that correlate with protection in relevant animal
models and any nonclinical ICPs that have been identified (for example, from
trials that assess passive protection and active immunization). This approach
may be the only option available for interpreting immune responses to some
new candidate vaccines. Nevertheless, ICPs derived wholly from nonclinical
data should be viewed with caution and attempts should be made to obtain a
clinical ICP whenever the opportunity arises (for example, when the vaccine is
used in the context of an outbreak).
If conducted, human challenge trials may also provide preliminary
evidence supporting an ICP. If a human challenge trial suggests a correlation
between a specific immunological parameter and protection, this may be further
WHO Technical Report Series, No. 1004, 2017
at each trial site who are not otherwise involved in the trial should administer
the products. If this is not feasible, or if the vaccines to be compared are
given by different routes or according to different schedules, attempts should
be made to maintain blinding of the trial site staff conducting the study visits
and assessments.
In trials intended to provide only descriptive analyses of the
immunogenicity data the trial sample size is usually based on considerations
of feasibility and collection of sufficient safety data to support the design of
sequential trials. Trials that aim to assess superiority or non-inferiority between
vaccine groups should be sized according to the intended power and the
predefined margins. It is recommended that protocols and statistical analysis
plans for each trial should be developed in conjunction with an appropriately
experienced statistician.
5.5.2.1 End-points
The choice of the primary trial end-point and the range of other end-points for
immunogenicity trials should take into account sections 5.2, 5.3 and 5.4 above.
Protocols should predefine the primary, co-primary, secondary and any other
end-points (which may be designated tertiary or exploratory). Co-primary end-
points may be appropriate in some cases, namely:
■■ The vaccine is intended to protect against multiple subtypes of the
same microorganism (for example, HPV vaccines or pneumococcal
conjugate vaccines).
■■ The vaccine contains multiple microorganisms (such as measles,
mumps and rubella vaccine) or multiple antigens (such as
combination vaccines used for the primary immunization series
in infants).
WHO Technical Report Series, No. 1004, 2017
type b and Neisseria meningitidis group C vaccines). In such cases the clinical
development programme should usually incorporate an assessment of immune
responses to a post-primary dose.
If it is not known whether post-primary doses of a new candidate
vaccine will be needed to maintain protection, it is preferable that this should
be determined from long-term follow-up of subjects who were enrolled in
efficacy trials and/or from post-licensure effectiveness studies. Although the
long-term monitoring of antibody persistence is important, these data alone
cannot determine if another dose is needed unless there is evidence, or a strong
reason to expect, that failure to maintain circulating antibody above a certain
level (for example, above the ICP if there is one) is associated with a risk of
breakthrough disease.
If it is unclear whether additional doses are needed it is prudent to
plan to obtain data on the immune response to doses administered at different
intervals after the last dose of the primary series so that such data are available
should it become clear that a further dose is required.
5.6.2.1.1 Modifying the use of the vaccine for which efficacy has been estimated
As described in section 6 below, vaccine efficacy trials are usually conducted in
specific target populations – characterized by factors such as age, region (which
may define the endemicity of some infectious diseases) and health status – using
the intended final vaccine posology. Before or after licensure, trials may be
conducted with the aim of extending the use of the vaccine to other populations
and/or to support alternative posologies.
When a different age group or posology is proposed it is usually very
clear that a bridging trial is necessary. A bridging trial may be required if there are
compelling scientific reasons to expect that the immune response to the vaccine,
and therefore its efficacy, could be significantly different to that documented
in a prior efficacy trial because of host factors (such as common underlying
conditions that may affect immune responses) and/or geographical factors (such
as distribution of subtypes of organisms and levels of natural exposure). In infants
there is also the possibility that very different levels of maternal antibody could
occur in different regions, resulting in variable interference with infant immune
responses to the primary series.
The trial design may involve a direct comparison between: (a) the new
posology and that used in the efficacy trial; or (b) the new intended population
and a control group consisting of subjects who are representative of the prior
efficacy trial population. It may also be acceptable to make an indirect (cross-
trial) comparison with the immunogenicity data that were obtained during the
efficacy trial.
The vaccine formulation and assay used should be the same as those
used in the efficacy trial whenever possible:
■■ If the exact vaccine used in the efficacy trial is no longer available
the comparator should be as similar as possible to the original
vaccine that was evaluated. Over time, it may be that the only bridge
back to the efficacy data is via a comparison with a licensed vaccine
that was itself licensed on the basis of a bridging efficacy trial. As the
number of bridging steps that have occurred between the original
efficacy data and the licensed comparator vaccine increases, the
reliance that may be placed on a demonstration of non-inferiority to
predict efficacy is weakened. This consideration also applies when
the vaccine for which efficacy was estimated contained a certain
number of subtypes but was later replaced by a vaccine containing
a larger number of subtypes on the basis of comparing immune
responses to the shared subtypes.
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■■ If the assay has changed and has not been, or cannot be, directly
compared to the original assay used during the efficacy trial it may
be possible to re-assay stored sera collected during the prior efficacy
trial in parallel with the sera from the new trial population.
If it remains unknown which immunological parameter best correlates
with efficacy it is preferable that the primary comparison between vaccines is
based on functional antibody whenever this is feasible.
construct (for example, whether the vaccine contains a live organism that is
replication competent), whether pregnant women can reasonably avoid exposure
to an infectious agent (for example, by not travelling) and whether they may
have the same risk of exposure but a greater risk of experiencing severe disease
compared to non-pregnant women of the same age.
Not all vaccines are, or need to be, evaluated in trials in pregnant women.
If there is no or very limited experience of the use of a vaccine in pregnant
women, NRAs may consider whether nonclinical data and any data available
from the clinical use of the vaccine and very similar vaccines could be provided
in the prescribing information.
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upon the main features of the design of the trials before initiation (including the
sample size) so that, subject to promising results, the data may be considered
robust and sufficient.
The availability of a licensed vaccine has potentially important
implications for the acceptability and feasibility of initiating or completing
additional efficacy trials that include a control group that does not receive active
vaccination. These issues should be discussed between NRAs and sponsors so
that expectations for the completion of additional efficacy trials are agreed upon
prior to the start of trials that could potentially support licensure.
If adequate data are not already available from public health authorities
then sponsors may have to conduct feasibility assessments in order to accurately
ascertain the clinical disease rates in various age subgroups of populations
before selecting trial sites. Any nationally recommended non-vaccine-related
preventive measures that are in place (for example, prophylactic drug therapy
in high-risk settings or in individuals at high risk, or the use of insect repellents
and bednets) should be identified. Trials are usually conducted against a
background of such measures.
Trial sites need to be sufficiently accessible to allow regular visits for
monitoring. Prior to initiation of the trial, sponsors may have to engage in site
capacity-building exercises, including training of study personnel, and may need
to provide essential infrastructure to support the trial (for example, adequate
blood-collection and processing facilities, refrigeration facilities suitable for the
vaccine and/or sera, access to competent laboratories, data-handling capacity
and communication methods to allow for electronic randomization schemes,
rapid reporting of safety data and other trial issues to the sponsor).
6.3.3.1 Control groups not vaccinated against the infectious disease to be prevented
Following consultation between the sponsor, NRA, ethics committees, local
public health authorities and investigators it may be appropriate to use a control
group that is not vaccinated against the disease to be prevented by the new
candidate vaccine. For example, this may be the case when the trial is to be
conducted in countries in which:
■■ no vaccine is yet licensed for prevention of the disease in question;
and/or
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In this design the primary analysis is conducted once a pre-specified total
number of cases has been detected – based, in a double-blind setting, on the
anticipated numbers in test and control groups required to demonstrate the
projected vaccine effect.
Alternative designs that allow for comparison with a control group that
is not vaccinated against the disease to be prevented may, at least in the short
term, include the following:
■■ In a randomized stepped wedge trial, the candidate vaccine
is administered to predefined groups in a sequential fashion.
Each predefined group is a unit of randomization. These may be
geographical groups or groups defined by host factors (for example,
age) or other factors (for example, attendance at a specific school
or residence within a specific health-care facility catchment area).
Such a design may be chosen when there is good evidence to
indicate that the vaccine will do more good than harm (affecting the
equipoise associated with randomization to a control group that is
not vaccinated against the disease to be prevented) and/or when it is
impossible to deliver the intervention to all trial participants within
a short time frame.
■■ In a ring vaccination trial, the direct contacts (and sometimes
secondary contacts) of a case may be randomized to vaccine or
control or may be randomized to receive immediate vaccination or
vaccination after a period of delay (21). This type of post-exposure
cohort trial usually requires smaller sample sizes than prospective
randomized controlled trials.
Ring vaccination trials may be particularly applicable when
the infectious disease to be prevented is associated with a relatively
WHO Technical Report Series, No. 1004, 2017
Clinical efficacy is usually assessed in the total randomized trial population (that
is, those who are assigned to receive vaccine and/or control) and in predefined
subsets of the randomized population.
The predefined trial populations should include as a minimum:
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7. Safety
7.1 General considerations
All clinical trials that are conducted pre-licensure or post-licensure should
include an exploration of safety.
The assessment of safety may be the primary objective, a co-primary
objective or a secondary objective in a clinical trial. Since the methods for
collection, analysis and interpretation of safety data during clinical trials contrast
with those applicable to post-licensure routine safety surveillance they are
considered separately below.
In principle, many of the approaches to documenting and reporting safety
data during vaccine clinical trials and conducting vaccine pharmacovigilance
activities are similar to those used for all medicinal products. The sections
that follow should be read in conjunction with the extensive guidance that
is available from numerous publications, and on the websites of WHO, the
Council for International Organizations of Medical Sciences (CIOMS), ICH
and individual regulatory bodies. The focus of the following sections is thus on
a number of methods and practices that are different for vaccines compared to
other medicinal products, and on issues that may need to be addressed because
of vaccine composition.
WHO Technical Report Series, No. 1004, 2017
infants will not be wholly applicable to toddlers and older subjects) and by the
route of administration (for example, nausea and vomiting could be solicited
symptoms for vaccines given orally). Fever should be documented using digital
thermometers and should be determined at a specific site (for example, rectal
or axillary in infants). Recordings of fever should be made at predefined times
and for a specified number of days after each dose. For subjective symptoms (for
example, fatigue and myalgia) a simple scoring system should be included in the
diaries to allow for the grading of severity.
Any self-administered treatments used to address signs or symptoms
(such as antipyretic and analgesic medicines) and any contact with – or treatment
administered by – a health-care professional should be captured. Instructions
on the use of antipyretics and analgesics should be stated in the clinical trial
protocol. If at the time of each dose a supply of a specific antipyretic or analgesic
was provided for use as needed, or as instructed in accordance with the protocol,
the post-dose usage recorded should be checked against returned supplies. If
prior safety data suggest that pre-vaccination antipyretic use is appropriate then
this can be administered and recorded by trial staff at the vaccination visit.
At each trial visit, whether involving face-to-face or telephone contact
between the trial subject/caregiver and site staff, all diary cards completed by
vaccinees or caregivers should be checked for level of completion and further
instructions given as needed to improve data recording after the next dose is
given. At face-to-face visits the prior vaccination site(s) should be inspected
for any remaining signs such as induration. Trial subjects or caregivers should
also be asked about the maximum extent of signs (for example, to determine
whether whole limb swelling occurred). Any unresolved local or systemic signs
and symptoms should be recorded and action taken as appropriate.
For all AEs that meet the criteria for classification as SAEs there should be
careful documentation of dates of onset, underlying conditions and concomitant
medications, and adequate follow-up to record the outcomes.
7.2.3
7.2.3.1 Causality
Section 8.5 of the WHO Global manual on surveillance of adverse events
following immunization (23) recommends that in clinical trials the investigator
should make a judgement on relatedness to vaccination for all solicited signs
and symptoms, and unsolicited AEs. The sponsor may have access to additional
information that is not available to investigators and should assess causality for
all SAEs. The assessment of relatedness to vaccination should take into account
factors such as:
7.2.3.2 Severity
Sufficient data should be collected for each solicited sign and symptom and
unsolicited AE in order to assess severity. Wherever possible, widely used grading
scales (including scales that may be age specific) should be used. The same scales
should be applied throughout the clinical development programme.
group that receives a licensed vaccine that does not have a similar
composition to the candidate vaccine. Any marked differences
between a candidate vaccine and a licensed vaccine that has the
same or very similar composition are generally not anticipated and
may require further investigation.
■■ Differences between clinical trials that may be observed in one or
both of the candidate vaccine and control groups for total or specific
AE reporting rates. It is important to consider possible explanations,
taking into account whether or not the same effect on the pattern
of reporting rates was observed in groups that received candidate
vaccines and licensed vaccines and whether the study was double-
blind or open-label. There may be real and anticipated differences
in vaccine reactogenicity between trial populations (for example,
age-related differences for specific AEs, such as higher fever rates in
trials conducted in infants and toddlers compared to trials in older
children and adults). When there is no clear explanation for the
differences observed, further investigation is merited. For example,
there may have been incomplete reporting of AEs or data-entry
errors, as well as cultural factors that lead to a greater reluctance to
report side-effects in some regions.
The number that would provide a 95% chance of observing one instance of an AE that occurs on average
1
Licence holders should demonstrate that they have adequate capability and
appropriate staff to collect, interpret and act upon the safety data received. It
is important that efforts are made to accurately identify the vaccine(s) and lot
number(s) associated with each AEFI report.
It has become routine at the time of licensure for detailed proposals to
be in place for post-licensure safety surveillance activities, often in the form
of risk‑management plans. These documents and proposals are then routinely
updated at intervals in line with additional data that become available. The
plans usually outline the safety specification for the vaccine on the basis of all
available safety data at the time of submitting each version of the plan, along
with details of routine and proposed additional pharmacovigilance and risk-
minimization activities.
When planning pharmacovigilance activities for a vaccine it is
important to take into account that, in addition to routine pharmacovigilance
(that is, passive surveillance), important information may come from other
sources, namely:
■■ Data from active safety surveillance, which may be put in place by
public health bodies when a vaccine is introduced into a national
routine immunization programme, or when the use of a vaccine
within a programme changes significantly (for example, an
entirely different age group is vaccinated for the first time). Active
surveillance seeks to ascertain completely the number of AEs in
persons given a dose of a vaccine using a pre-organized process. It
may involve reviewing medical records or interviewing patients and/
or physicians in a sample of sentinel sites to ensure that complete
and accurate data are collected on reported AEs from those sites.
■■ Large databases that link information on vaccination history in
patient records with the occurrence of specific types of illness. These
WHO Technical Report Series, No. 1004, 2017
was then held in Geneva, Switzerland, 3 May 2016 and was attended by the
following participants: Dr G. Coleman, Health Canada, Canada; Dr M. Darko,
Food and Drugs Authority, Ghana; Dr D. Etuko, National Drug Authority,
Uganda; Dr E. Griffiths, Consultant, Kingston-upon-Thames, England; Dr S.
Kennedy, University of Liberia, Liberia; Dr J. McEwen, Therapeutic Goods
Administration, Australia; Dr M. Powell, Medicines and Healthcare products
Regulatory Agency, England; Dr R. Sheets, Consultant, Silver Spring (MD), the
USA; Dr J. Southern, Medicines Control Council, South Africa; Dr Y. Sun, Paul-
Ehrlich-Institut, Germany; Dr K. Zoon, National Institutes of Health, the USA;
and Dr I. Knezevic, World Health Organization, Switzerland.
Based on the comments received during the public consultation and
on the discussions of the above Working Group meeting, the document WHO/
BS/2016.2287 was prepared by the above-mentioned WHO drafting group.
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The document was then posted on the WHO Biologicals website for
a third round of public consultation from 27 July to 16 September 2016 and
comments received from: Dr B. Brock, Sanofi Pasteur, the USA (provided the
consolidated comments of the IFPMA); Dr M. Cavaleri, European Medicines
Agency, England; Dr G. Coleman, Health Canada, Canada; Dr D. Kim and
Dr M. Jin, Ministry of Food and Drug Safety, Republic of Korea; Dr A.W. Lee,
Dr A. Sitlani and Dr W. Straus, Merck & Co., the USA; Dr T. Lu, Therapeutic
Goods Administration, Australia; Dr S.A. Nishioka, Ministry of Health, Brazil;
Office of International Affairs, Instituto Nacional de Vigilancia de Medicamento,
Colombia; Dr S-C Shin, Green Cross Corporation, Republic of Korea; and
Dr Y. Sun, Paul-Ehrlich-Institut, Germany. Dr Noni MacDonald, DalHousie
University, Halifax, Canada and Dr Anna Taddio, University of Toronto, Toronto,
Canada, provided comments on the safety evaluation, particularly on the pain
of injection.
Further changes were subsequently made to document WHO/BS/
2016.2287 by the WHO Expert Committee on Biological Standardization.
References
1. Guidelines on clinical evaluation of vaccines: regulatory expectations. In: WHO Expert Committee
on Biological Standardization: fifty-second report. Geneva: World Health Organization; 2004:
Annex 1 (WHO Technical Report Series, No. 924; http://www.who.int/biologicals/publications/trs/
areas/vaccines/clinical_evaluation/035-101.pdf?ua=1, accessed 4 February 2017).
2. WHO guidelines on nonclinical evaluation of vaccines. In: WHO Expert Committee on Biological
Standardization: fifty-fourth report. Geneva: World Health Organization; 2005: Annex 1 (WHO
Technical Report Series, No. 927; http://www.who.int/biologicals/publications/trs/areas/vaccines/
nonclinical_evaluation/ANNEX%201Nonclinical.P31-63.pdf?ua=1, accessed 4 February 2017).
3. Guidelines for good clinical practice (GCP) for trials on pharmaceutical products. In: WHO Expert
Committee on the Use of Essential Drugs: sixth report. Geneva: World Health Organization; 1995:
Annex 3 (WHO Technical Report Series, No. 850; http://whqlibdoc.who.int/trs/WHO_TRS_850.
pdf?ua=1, accessed 4 February 2017).
4. WHO good manufacturing practices for pharmaceutical products: main principles. In: WHO Expert
Committee on Specifications for Pharmaceutical Preparations: forty-eighth report. Geneva:
World Health Organization; 2013: Annex 2 (WHO Technical Report Series, No. 986; http://www.
who.int/medicines/areas/quality_safety/quality_assurance/TRS986annex2.pdf?ua=1, accessed
4 February 2017).
5. WHO good manufacturing practices for biological products. In: WHO Expert Committee on
Biological Standardization: sixty-sixth report. Geneva: World Health Organization; 2016: Annex 2
(WHO Technical Report Series, No. 999; http://www.who.int/biologicals/areas/vaccines/Annex_2_
WHO_Good_manufacturing_practices_for_biological_products.pdf?ua=1, accessed 27 March
2017).
6. Guidelines on the nonclinical evaluation of vaccine adjuvants and adjuvanted vaccines. In:
WHO Expert Committee on Biological Standardization: sixty-fourth report. Geneva: World
Health Organization; 2014: Annex 2 (WHO Technical Report Series, No. 987; http://www.who.int/
biologicals/areas/vaccines/TRS_987_Annex2.pdf?ua=1, accessed 4 February 2017).
571
WHO Expert Committee on Biological Standardization Sixty-seventh report
7. Guidelines on procedures and data requirements for changes to approved vaccines. In: WHO Expert
Committee on Biological Standardization: sixty-fifth report. Geneva: World Health Organization;
2015: Annex 4 (WHO Technical Report Series, 993; http://www.who.int/biologicals/vaccines/
Annex4_Guidelines_changes_to_approved_vaccines_eng.pdf?ua=1, accessed 4 February 2017).
8. Guidelines for independent lot release of vaccines by regulatory authorities. In: WHO Expert
Committee on Biological Standardization: sixty-first report. Geneva: World Health Organization;
2013: Annex 2 (WHO Technical Report Series, No. 978; http://www.who.int/biologicals/TRS_978_
Annex_2.pdf?ua=1, accessed 4 February 2017).
9. Recommendations for the evaluation of animal cell cultures as substrates for the manufacture
of biological medicinal products and for the characterization of cell banks. In: WHO Expert
Committee on Biological Standardization: sixty-first report. Geneva: World Health Organization;
2013: Annex 3 (WHO Technical Report Series, No. 978; http://www.who.int/biologicals/vaccines/
TRS_978_Annex_3.pdf?ua=1, accessed 4 February 2017).
10. Clinical considerations for evaluation of vaccines for prequalification. Points to consider for
manufacturers of human vaccines. Geneva: World Health Organization; 2010 (http://www.
who.int/immunization_standards/vaccine_quality/clinical_considerations_oct10.pdf, accessed
4 February 2017).
11. Immunization in practice: a practical guide for health staff. 2015 update. Geneva: World
Health Organization; 2015 https://extranet.who.int/iris/restricted/bitstream/10665/193412/1/
9789241549097_eng.pdf, accessed 4 February 2017).
12. Expert consultation on the use of placebos in vaccine trials. Meeting report. Geneva: World Health
Organization; 2013 (http://apps.who.int/iris/bitstream/10665/94056/1/9789241506250_eng.pdf,
accessed 4 February 2017).
13. Plotkin SA, Gilbert PB. Nomenclature for immune correlates of protection after vaccination.
Clin Infect Dis. 2012;54(11):1615–7 (https://academic.oup.com/cid/article/54/11/1615/321882/
Nomenclature-for-Immune-Correlates-of-Protection?related-urls=yes&legid=cid;cis238v1,
accessed 27 March 2017).
14. The importance of pharmacovigilance: safety monitoring of medicinal products. Geneva: World
Health Organization; 2002 (http://apps.who.int/medicinedocs/pdf/s4893e/s4893e.pdf, accessed
4 February 2017).
15. Siegrist C-A. Vaccine immunology. In: Plotkin SA, Orenstein WA, Offit PA, editors. Vaccines, sixth
edition. Philadelphia (PA): Elsevier Saunders; 2012, chapter 2.
WHO Technical Report Series, No. 1004, 2017
16. ICH tripartite guideline Q2(R1). Validation of analytical procedures: text and methodology (Step
4 version) Geneva: International Conference on Harmonisation of Technical Requirements for
Registration of Pharmaceuticals for Human Use; 1996; 6–13 (http://www.ich.org/fileadmin/
Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__Guideline.pdf,
accessed 4 February 2017).
17. Recommendations to assure the quality, safety and efficacy of DT-based combined vaccines. In:
WHO Expert Committee on Biological Standardization: sixty-third report. Geneva: World Health
Organization; 2014: Annex 6; Table 6.2 (WHO Technical Report Series, No. 980; http://www.who.
int/biologicals/vaccines/Combined_Vaccines_TRS_980_Annex_6.pdf?ua=1, accessed 27 March
2017).
18. Buttery JP, Riddell A, McVernon J, Chantler T, Lane L, Bowen-Morris J et al. Immunogenicity
and safety of a combination pneumococcal-meningococcal vaccine in infants: a randomized
controlled trial. JAMA. 2005;293(14):1751–8 (http://jamanetwork.com/journals/jama/fullarticle/
200691, accessed 4 February 2017).
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Annex 10
Human challenge trials for vaccine development:
regulatory considerations
1. Introduction 578
2. Background 580
3. Purpose and scope 580
4. Purposes of human challenge trials in vaccine development 580
5. Study design of human challenge trials 582
6. Operational aspects 583
7. Some key ethical considerations 584
Authors and acknowledgements 585
References 587
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Abbreviations
GCP good clinical practice
GMO genetically modified organism
ICP immune correlate of protection
NRA national regulatory authority
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1. Introduction
Infectious human challenge trials involve the deliberate exposure of human
volunteers to infectious agents. Trial participants are intentionally challenged
(whether or not they have been vaccinated) with an infectious disease organism.
This challenge organism may be close to wild-type and pathogenic, adapted
and/or attenuated from wild-type with less or no pathogenicity, or genetically
modified in some manner.
Human challenge trials have been conducted over hundreds of years
and have contributed vital scientific knowledge that has led to advances in the
development of drugs and vaccines. Nevertheless, such research can appear to
be in conflict with the guiding principle in medicine to do no harm. A number
of well-documented historical examples of human exposure studies would be
considered unethical by current standards. It is essential that challenge trials be
conducted within an ethical framework in which truly informed consent is given.
When conducted, human challenge trials should be undertaken with abundant
forethought, caution and oversight. The value of the information to be gained
should clearly justify the risks to human subjects.
Although human challenge trials are not a required element of every
vaccine development programme, there are many reasons why a developer may
ask to conduct a “challenge-protection” study with humans, which might normally
be conducted in animals. Animal models are often quite imprecise in reflecting
human disease, and many infectious organisms against which a developer might
wish to develop a vaccine are species-specific for humans. Human challenge
trials may be safely and ethically performed in some cases, if properly designed
and conducted. Considerable insight can then be gained into the mode of action
and potential benefit of drugs and vaccines in humans. However, there are also
limitations on what challenge trials may be able to ascertain because, as with
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disease an organism causes has an acute onset, can be readily and objectively
detected, and existing efficacious treatments (whether curative or palliative) can
be administered at an appropriate juncture in disease development to prevent
significant morbidity and eliminate mortality.
It will also be important to consider the regulatory framework in which
the human challenge trial may be conducted. In some countries, challenge stocks
are expected to be regulated in the same manner as vaccines, and are expected
to be studied with authorization in accordance with clinical trial regulations,
whether or not an investigational vaccine is to be used in the same clinical
investigation protocol. For example, a challenge trial might be conducted to titrate
the challenge organism in humans (before using the challenge in a vaccine study)
in order to determine the proper dose of the challenge organism to administer,
and to characterize the symptoms, kinetics, shedding and transmissibility to be
expected from the challenge. The dose of challenge organism is usually titrated to
induce a relatively high attack rate while limiting disease severity. In cases where
the challenge should be studied in compliance with clinical trial regulations
there is greater clarity about regulatory expectations, including the quality of the
challenge stock to be used, because the clinical trial regulations or requirements
would apply. However, in many countries, because the challenge stock is not itself
considered to be a medicinal product, such characterization/model development
studies would not come under national regulatory authority (NRA) review and
authorization. Thus, much less clarity would exist on regulatory expectations and
issues of quality in such cases.
It should be understood that a pathogenic challenge strain will not have
the “safety” of an intended safe candidate vaccine. However, its quality should
be comparable to a candidate vaccine at the same clinical trial phase. Ideally,
a human challenge trial to establish the challenge model (that is, without use
of an investigational medicinal product) should also match the expectations
for conducting a vaccine study – that is, compliance with good clinical
practice (GCP) and subject to approval or concurrence under a Clinical Trial
Authorization by NRAs and ethics committees on the basis of requirements
appropriate for this type of study. If such a framework does not exist, countries
are encouraged to establish an appropriate regulatory and ethical framework for
challenge trials. However, there may be no regulatory framework to promulgate
such expectations in the country where the challenge study is to be conducted.
Trial sponsors, vaccine developers, researchers and other involved parties should
determine what regulatory expectations the relevant NRA may have when clarity
does not exist and when the human challenge study is intended to support the
development of a vaccine candidate they would ultimately like to license (that is,
obtain marketing authorization or registration).
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2. Background
In July 2014, WHO held a consultation on Clinical evaluation of vaccines:
regulatory expectations (2). One area that was considered to be an important
element in facilitating vaccine development was human challenge trials. It was
recognized that the regulation of such trials needed to be well-defined by NRAs
and that vaccine developers and manufacturers needed to be aware of regulatory
expectations in this area.
This WHO guidance document on human challenge trials should be
read in conjunction with the updated WHO Guidelines on clinical evaluation of
vaccines: regulatory expectations (3) which were adopted, along with the current
document, by the WHO Expert Committee on Biological Standardization in
October 2016.
in vaccine development
Human challenge trials are considered as a model by which challenge protection
can be evaluated and represent one possible approach for vaccine development.
Therefore, all principles for the clinical evaluation of vaccines should
apply, including the need for approval by the NRA and ethical committees as
well as compliance to GCP.
A vaccine developer may conduct human challenge trials to accomplish
one or more aims. The aims of the study determine the clinical phase in which the
study is conducted. Human challenge trials are often a type of efficacy-indicating
study, but most would not be considered to be pivotal efficacy studies. Almost all
would be pilot in nature and performed to gain useful information to aid in the
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1
When the timeline of vaccine development is viewed as a graph from early to the left to late to the
right, shifting the risk of failure earlier (left) in the timeline could: (a) minimize risk to human subjects by
avoiding large efficacy studies of vaccines that would not prove efficacious; (b) result in significant cost
and resource savings; and (c) minimize lost opportunity costs by abandoning an unpromising candidate
before committing greater expenditures to higher-phase clinical trials.
2
The target population in a particular country may have a higher rate of individuals with, for example,
sickle cell trait, poorer nutritional status or greater parasitic load in “normal” flora – any of which might
affect immune responsiveness in the endemic setting and thus efficacy (benefit) compared to the efficacy
trial population (ideal setting) or safety (greater risks). Either of these would have an impact on the risk–
benefit decision-making.
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WHO Expert Committee on Biological Standardization Sixty-seventh report
This might entail a challenge study in adults to extrapolate when children might need booster doses.
3
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Annex 10
evidence upon which regulators could take a clinical trial or licensure decision.
Thus, the purpose of the study would influence its design, which would in
turn influence the conclusions and decisions that might be made by regulators
following consideration of the study results.
6. Operational aspects
In addition to general principles for all clinical trials in human subjects there are
some unique and important operational aspects to consider when conducting
a human challenge trial. Human challenge trials should be undertaken in
accordance with a protocol, and in special facilities that are designed and operated
in a manner that prevents the spread of the challenge organism to people outside
the study or to the environment. These clinical facilities should be capable of
providing continuous monitoring and medical attention at the appropriate
point(s) in time after the challenge is given. In addition to providing immediate
access to appropriate medical care and treatment, the facilities should be designed
to prevent the spread of disease, particularly when the challenge organism is a
GMO or an organism that is not endemic to the locality. These facilities may
need to be operated in a manner that permits all waste (including excrement) to
be collected and decontaminated before release. All staff, including janitorial and
administrative staff, might be required to work in personal protective equipment
appropriate for the pathogenicity of the challenge organism and its potential
hazard to the environment, and should be informed of the potential risks. It
should be noted that not all human challenge trials require such a high level of
control. When the challenge organism is attenuated and the wild-type organism
is likely to be present in the locality anyway, it may be adequate to conduct
human challenge trials in an outpatient setting or with appropriate procedures
to prevent spread. Examples of such approaches and procedures include the
use of BCG vaccine as a challenge organism, the use of bandaging to cover and
prevent spread from an intramuscular injection (assuming the organism is not
shed by other means) and the use of malaria challenge during winter months in a
temperate region. There may be other circumstances in which a human challenge
trial is undertaken, for example where the target organism of the vaccine to be
developed is not present in the location where the target group for its indication
lives (for example, in case of a traveller vaccine) – when the risk of spread of
the organism is low, human challenge trials using appropriate procedures could
be undertaken.
It may be necessary to ensure that controls and vaccinees are housed
together if an objective of the human challenge trial is to identify the potential
for transmissibility. In such a situation, only the vaccinees or unvaccinated
participants would be challenged, and the controls (who were not challenged)
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characteristics.
The ethical considerations concerning challenges in clinical trials
should be thoroughly evaluated. During a WHO Expert Consultation held in
January 2013 consideration was given to the way in which ethical principles
should be applied to vaccine trials. The main consultation topic concerned the
use of placebo in such trials, and a set of considerations for NRAs and ethics
committees was provided in the meeting report (4) and subsequently published
recommendations (5). Although specifically intended to facilitate review of
the proposed use of placebo in vaccine trials on a case-by-case basis these
considerations and recommendations are likely to have applicability to human
challenge trials.
It has to be acknowledged that in reality some individuals are greater
risk-takers than others, and that those who are risk averse would be unlikely to
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accept the risk of receiving a challenge. The key to asking individuals to accept
the risk from a challenge study (in which they have little potential to receive
individual benefit) lies in the element of informed consent. Healthy adults may
consent when they are well informed and understand what the risks are that
they are agreeing to take – even if those risks may be considerably greater than
minimal (for example, accepting that they will develop an acute, but manageable,
disease that will resolve but in the meantime may cause considerable morbidity,
such as severe diarrhoea managed with fluid and electrolyte replacement). There
could be some potential for direct benefit should the trial participant become
immune to the disease caused by the challenge (or wild-type) organism but,
conversely, pre-existing immunity upon exposure to the wild-type organism in
the future may be harmful. Thus, in appropriate situations, it may be considered
ethical to ask healthy and informed adults to consent to volunteer and participate
in a human challenge trial whether they will receive an investigational vaccine
that may or may not protect them from the challenge organism, a placebo that
will not protect them or only the challenge organism itself. However, it is an
absolute requirement that accepting such risks and providing voluntary consent
are based upon being truly informed. For this reason (the absolute requirement
for truly informed consent) it is not deemed acceptable at this time to consider
conducting human challenge trials in children, or in any other vulnerable
population with diminished capacity to give informed consent. One possible
exception to this principle that might be considered would be a challenge model
that used a licensed live-attenuated vaccine as the challenge organism.
The need to minimize the risks to subjects in clinical trials calls for
investigators to give due consideration to whether the challenge organism needs
be pathogenic or not, or to what degree. As noted above, the aim or purpose of
the study may drive decisions on pathogenicity or attenuation, but the ethical
precept of minimizing risks to human subjects – to the maximum extent feasible
within the framework of sound science – should be given due consideration.
Key to such considerations is the credibility of the data to support regulatory
decision-making, which also needs to be taken into account when deciding how
pathogenic or attenuated the challenge organism needs to be.
Grimalkin Partners, the USA; and particularly the report of the conference
organized by the International Alliance for Biological Standardization on
Human Challenge Trials in October 2014, which served as an important source
of information during the preparation of this document.
The proposal to prepare WHO guidance on human challenge trials was
developed during a WHO Consultation on clinical evaluation of vaccines held in
Geneva, Switzerland, 17–18 July 2014, and attended by the following participants:
Dr P. Annunziato, Merck & Co., the USA; Dr N. Bhat, Program for Appropriate
Technology in Health, the USA; Dr A. Chatterjee, Biological E Ltd, India; Dr K.
Chirgwin, Bill & Melinda Gates Foundation, the USA; Dr G. Coleman, Health
Canada, Canada; Dr D. Tuan Dat, The Company for Vaccines and Biological
Production No. 1 (Vabiotech), Viet Nam; Dr P.E. Fast, International AIDS
Vaccine Initiative, the USA; Dr G. Foglia, Sanofi Pasteur, the USA; Dr M. Gruber,
United States Food and Drug Administration, Center for Biologics Evaluation
and Research, the USA; Dr P.M. Heaton, Bill & Melinda Gates Foundation,
the USA; Dr D. Kaslow, Program for Appropriate Technology in Health,
the USA; Dr Y.H. Lee, Ministry of Food and Drug Safety, Republic of Korea;
Dr D.J.M. Lewis, University of Surrey, England; Dr A. Lommel, Paul-Ehrlich-
Institut, Germany; Dr J. McEwen, Therapeutic Goods Administration, Australia;
Dr P. Neels, Vaccine-Advice BVBA, Belgium; Dr M. Nijs, GlaxoSmithKline
Biologicals, Belgium; Dr S.A. Nishioka, Ministry of Health, Brazil; Dr A. Podda,
Novartis Vaccines Institute for Global Health, Italy; Dr M. Powell, Medicines and
Healthcare products Regulatory Agency, England; Dr A. Ramkishan, Central
Drugs Standard Control Organization, India; Dr R. Sheets, Consultant, Silver
Spring (MD), the USA; Dr J. Shin, WHO Regional Office for the Western Pacific,
Philippines; Dr P. Smith, London School of Hygiene and Tropical Medicine,
England; Dr J. Southern, Medicines Control Council, South Africa; Dr Y. Sun,
Paul-Ehrlich-Institut, Germany; Dr Z. Yang, Center for Drug Evaluation, China;
and Dr U. Fruth, Dr I. Knezevic, Mr O.C. Lapujade, Dr V. Moorthy, Dr K. Vannice
WHO Technical Report Series, No. 1004, 2017
References
1. Sheets RL, Fritzell B, Aguado de Ros MT. Human challenge trials in vaccine development:
Strasbourg, September 29 – October 1, 2014. Biologicals. 2016;44(1):37–50 (abstract: http://www.
sciencedirect.com/science/article/pii/S104510561500113X, accessed 4 February 2017).
2. Knezevic I, Moorthy V, Sheets RL. WHO consultation on clinical evaluation of vaccines, 17–18 July
2014, WHO Headquarters, Geneva, Switzerland. Vaccine. 2015;33(17):1999–2003 (http://www.
sciencedirect.com/science/article/pii/S0264410X15001139, accessed 4 February 2017).
3. Guidelines on clinical evaluation of vaccines: regulatory expectations. In: WHO Expert Committee
on Biological Standardization: sixty-seventh report. Geneva: World Health Organization; 2017:
Annex 9 (WHO Technical Report Series, No. 1004).
4. Expert consultation on the use of placebos in vaccine trials. Meeting report. Geneva: World
Health Organization; 2013 (http://apps.who.int/iris/bitstream/10665/94056/1/9789241506250_
eng.pdf, accessed 4 February 2017).
5. Rid A, Saxena A, Baqui AH, Bhan A, Bines J, Bouesseau M-C et al. Placebo use in vaccine
trials: Recommendations of a WHO expert panel. Vaccine. 2014;32(37):4708–12 (http://www.
sciencedirect.com/science/article/pii/S0264410X14005374, accessed 4 February 2017).
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Annex 11
Biological substances: WHO International Standards,
Reference Reagents and Reference Panels
The provision of global measurement standards is a core normative WHO
activity. WHO reference materials are widely used by manufacturers, regulatory
authorities and academic researchers in the development and evaluation of
biological products. The timely development of new reference materials is crucial
in harnessing the benefits of scientific advances in new biologicals and in vitro
diagnosis. At the same time, management of the existing inventory of reference
preparations requires an active and carefully planned programme of work to
replace established materials before existing stocks are exhausted.
The considerations and guiding principles used to assign priorities
and develop the programme of work in this area have previously been set out
as WHO Recommendations.1 In order to facilitate and improve transparency
in the priority-setting process, a simple tool was developed as Appendix 1 of
these WHO Recommendations. This tool describes the key considerations taken
into account when assigning priorities, and allows stakeholders to review and
comment on any new proposals being considered for endorsement by the WHO
Expert Committee on Biological Standardization.
A list of current WHO International Standards, Reference Reagents and
Reference Panels for biological substances is available at: http://www.who.int/
biologicals.
At its meeting in October 2016, the WHO Expert Committee on
Biological Standardization made the changes shown below to the previous list.
Recommendations for the preparation, characterization and establishment of international and other
1
biological reference standards (revised 2004). In: WHO Expert Committee on Biological Standardization:
fifty-fifth report. Geneva: World Health Organization; 2006: Annex 2 (WHO Technical Report Series, No. 932;
http://www.who.int/immunization_standards/vaccine_reference_preparations/TRS932Annex%202_
Inter%20_biol%20ef%20standards%20rev2004.pdf?ua=1, accessed 4 March 2017).
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WHO Expert Committee on Biological Standardization Sixty-seventh report
Additions2
Preparation Activity Status
Blood products and related substances
Ancrod 54 IU/ampoule Second WHO
International Standard
Batroxobin 50 U/ampoule First WHO Reference
Reagent
Blood coagulation factor FXI:C = 0.71 IU/ampoule Second WHO
XI (plasma, human) FXI:Ag = 0.78 IU/ampoule International Standard
Thromboplastin 1.11 IU/mL Fifth WHO International
(recombinant, human, Standard
plain)
Thromboplastin (rabbit, 1.21 IU/mL Fifth WHO International
plain) Standard
In vitro diagnostics
Zika virus RNA for 50 000 000 IU/mL First WHO International
NAT‑based assays* Standard
Ebola virus VP40 antigen Panel containing low- and First WHO Reference
medium-titre VP40 samples Panel
plus negative sample; no
unitage assigned
Dengue virus serotypes Four separate reference First WHO reference
1–4 RNA for NAT-based reagents with the following reagents
assays** assigned values:
DENV-1 RNA = 13 500 units/mL;
WHO Technical Report Series, No. 1004, 2017
DENV-2 RNA = 69 200 units/mL;
DENV-3 RNA = 23 400 units/mL;
DENV-4 RNA = 33 900 units/mL
Hepatitis B virus DNA 5.98 log10 IU/mL Fourth WHO
for NAT-based assays International Standard
Unless otherwise indicated, all materials are held and distributed by the National Institute for Biological
2
Standards and Control, Potters Bar, Herts, EN6 3QG, England. Materials identified by an * in the above list
are held and distributed by the Paul-Ehrlich-Institut, 63225 Langen, Germany. Materials identified by an **
in the above list are held and distributed by the Center for Biologics Evaluation and Research, Food and
Drug Administration, Silver Spring, MD 20993, the USA.
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591
SELECTED WHO PUBLICATIONS OF RELATED INTEREST
Further information on these and other WHO publications can be obtained from
WHO Press, World Health Organization, 1211 Geneva 27, Switzerland
(tel.: +41 22 791 3264; fax: + 41 22 791 4857; email: [email protected];
order online: www.who.int/bookorders)
This report presents the recommendations of a WHO Expert
Committee commissioned to coordinate activities leading to the
adoption of international recommendations for the production
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W H O Te c h n i c a l R e p o r t S e r i e s
and control of vaccines and other biological substances, and the
establishment of international biological reference materials.
Following a brief introduction, the report summarizes a number
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of general issues brought to the attention of the Committee. The